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United States 
Environmental Protection 
Agency 


Environmental Research 
Laboratory 
South Ferry Road 
Narragansett, Rl 02882 


Research & Development ./ __ 

Advances in Marine 
Environmental Research 


Proceedings 
of a Symposium 














E PA- 600/9-79-035 
September 1979 


Advances in Marine 
Environmental Research 

Proceedings of a 
Symposium 


Francine Sakin Jacoff, 
Editor 


Environmental Research Laboratory 
Office of Research and Development 
U.S. Environmental Protection Agency 
Narragansett, Rhode Island 02882 


DISCLAIMER 


QH SHI 

.<r 

. ^3 A 3 ? 


This report has been reviewed by the Narragansett Environental 
Research Laboratory, U.S. Environmental Protection Agency, 
and approved for publication. Mention of trade names or 
commercial products does not constitute endorsement or 
recommendation for use. 



Sq -(p CO 5 ^ ) 




11 


DEDICATION 


ADVANCES IN MARINE POLLUTION RESEARCH 


A decade ago, there were only a handful of scientists throughout the world 
engaged in the field of science called ecology. With rising social consciousness 
and an escalating series of local, national and global environmental problems, 
there was an outcry for the application of scientific analysis to these problems. 
The result of this was an evolution of a new field, called pollution research, 
which had as its cornerstone the science of ecology. 

This volume includes papers that will discuss many of the specific aspects of 
marine ecology and marine pollution research. You will find authors who are 
studying the transformation and movements of pollutants in chemical systems, 
as well as those who are attempting to miniaturize and model ecosystems with 
the microcosms. The papers contained herein are a benchmark of marine 
pollution research. 

We are dedicating the volume to one of the founders of modern ecology and 
marine pollution research, Eugene P. Odum. Dr. Odum has dedicated his life to 
understanding the holistic processes of ecosystems and man’s interaction with 
these complex biological, physical and chemical systems. His pioneering work 
in wetlands and radioecology led to his synthesized works in 
FUNDAMENTALS OF ECOLOGY. The more that we attempt to understand 
and unravel the complexities of modern marine ecosystems, the more we 
recognize that the basic principles expoused by Eugene Odum are true. Not 
only are we realizing that we cannot uncouple the various components of 
ecosystems, but that man himself is coupled into these complex systems. 

Do not read the volume with the expectation of understanding all the 
answers to major marine pollution problems today—but read it as a 
state-of-the-art document outlining our advances in a rapidly changing and 
evolving science. Throughout all the papers, attempt to follow Odum’s 
guidance, and to understand how the discussion of various parts of the problem 
can be combined into holistic concepts that have eluded us in the past. Our 
field of marine ecology has not evolved to that of a predictive science; we lack 
basic hypotheses and understandings to make it so. 

I hope that the reader will view these papers through Odum’s 
“macroscope”, and in this way gain insights into the holistic view of our oceans 
and coastal waters that will allow man to live to closer harmony with the sea. 

Eric D. Schneider, Director 
Environmental Research Laboratory 
Narraggasett, R.I. 


111 


KEYNOTE ADDRESS 


DEDICATION OF NEW WING OF 
NARRAGANSETT EPA LABORATORY 

JUNE 1977 

Delivered by 

Eugene P. Odum, Director 
Institute of Ecology, University of Georgia 

The theme of my address at today’s dedication is that the time has come to 
adopt a holistic approach to researching and managing our environmental 
problems. This is not to say that we abandon the traditional reductionist way 
of science which involves dividing up a complex problem into small 
components that are then assigned to specialists for detail study. Rather, we 
perhaps need to follow the general procedure we use in microscopy, namely, 
shifting back and forth between powers so as to examine the subject at 
different levels of organization. To put it another way, we need to develop the 
“macroscope” as a tool as well as the microscope. Most of all, we need to 
promote integrated team research as well as reward the individual effort that is 
the traditional, and too often the only, criterion for promotion in universities 
and research institutions. 

Reductionism in science has led to important discoveries in physics, 
chemistry, molecular biology and genetics, but this approach comes up short in 
ecology where the exciting problems, and also those of most concern to 
society, lie at the ecosystem level rather than at the molecular level. The 
Environmental Protection Agency was organized by society to fight cancer at 
the ecosystem level, not at the cell or organism level. Theories, and tools, must 
be organized accordingly, since procedures appropriate for one level of concern 
may not be appropriate at all for another level of study. 

Holism as a basic operational principle or paradigm rests on the theory of 
hierarchal systems, a theory not yet fully understood nor accepted by many 
scientists. Since there is both continuity and discontinuity in the evolution of 
the universe, development may be viewed as continuous because it is 
never-ending, but also discontinuous because it passes through a series of 
different levels of organization with vertical as well as horizontal integration. 
The keystone in the theory of hierarchal organization is the concept of 
emergent properties. As components, or subsets, are combined to produce 
larger functional wholes, new properties emerge that were not present or not 
evident at the next level below. In speaking of these matters in general lectures, 
I often use water as an example. Water has many unique properties not shared 


IV 


by the components, hydrogen and oxygen. To cite a few, it is a liquid, 
chemically inert, and has its maximum density of 4°C; in contrast to the two 
gaseous components having none of these characteristics. It is obvious that the 
holistic approach of studying water as water (as a whole molecular complex) 
would reveal these important integrative, or “emergent”, properties more easily 
and quickly than the reductionist approach keying on the study of the 
component parts. Thus, it would be very difficult, if not impossible, to deduce 
the maximum-density-at-4°C property of water from knowledge of the 
properties of hydrogen and oxygen as they occur in their separate states. 

Thus, the forest is indeed more than a collection of trees, to quote an old 
adage. As a specific example of emergent properties at the ecosystem level, I 
might cite the work my brother and I did on a coral reef on a Pacific Atoll, as 
was alluded to by Frank Lowman in his introduction. We measured the 
metabolism of the intact reef by monitoring oxygen changes in the water 
flowing over the reef. We also did a detailed trophic analysis as a means of 
charting major energy flows, and were able to construct an energy budget for 
the whole system. It became evident from these analyses that coral animals and 
associated algae were much more closely linked metabolically than had 
previously been supposed, and that the inflow of nutrients and animal food 
from surrounding ocean water was inadequate to support the reef community 
if corals and other biota were functioning in ordinary food chains. We 
theorized that the observed very high rate of productivity for the reef as a 
whole was an emergent property resulting from symbiotic linkages that 
maintain efficient energy use and nutrient recycling between autotrophic and 
heterotrophic components. I believe we can say that subsequent work on 
Pacific reefs has verified this hypothesis. 

As an interesting aside, we suggest that these coral reef discoveries have at 
least philosophical significance for urban-industrial man. The Pacific coral reef 
as an oasis in a desert ocean can stand as an object lesson for man who must 
now realize that mutalism between autotrophic and heterotrophic components, 
and between producers and consumers in the societal realm, coupled with 
efficient recycling of materials and use of energy, are the keys to maintaining 
prosperity in a world of limited resources. Only by moving up in our thinking, 
in our research, and in our management to the ecosystem level in the hierarchal 
system can we accomplish this vital mission. During the industrial revolution 
mankind essentially “uncoupled” himself from nature. Because the individual 
in industrial societies no longer is directly dependent on the natural 
environment for his day-to-day needs, he forgets how dependent we really are 
on natural processes that produce food, recycle water, purify air, and so on. 
Our food, for example, comes in on a long and complex chain of production, 
processing, and transportation so we are not really aware of where it came 
from or how much energy was expended, or how much pollution created, and 


v 


so on. It is definitely time to recouple the house of man and the house of 
nature and assess and manage them as one integrated ecosystem. 

In recent months the writing of environmental impact statements, as 
required by NEPA, has been criticized in the pages of Science and other 
professional magazines as being superficial and exercises of bureaucratic 
futility. As I see it, current impact assessment is not so much bad or inadequate 
science as it is wrong-level applied science, a viewpoint that has not been 
emphasized in recent discussions of the subject. In other words, if NEPA is to 
survive the economic and political pressure of the future, assessment must 
evolve as rapidly as possible from the present largely descriptive component 
approach to a more holistic approach which combines the use of broad 
ecosystem-level indices of structure and function with specific local or 
population factors (i.e., “red flags”) that are of special public concern (such as 
fish or game, or an endangered species). Also, economic and ecologic 
considerations must be integrated, not undertaken as separate studies without 
common denominators. This can be done, and if I had time I could describe 
two cases where we were successful in such a merger. (Write me and I’ll send 
reprints.) 


Finally, the impact-assessor and the decision-maker should be part of the 
same team, or at least sit around the same table to review all the alternatives. In 
other words, a good assessment cannot be made piecemeal any more than one 
can understand water or a coral reef by component study alone. 


So much for general theory; now for some suggestions for EPA and 
Directors of EPA laboratories. In pursuing its mission to reduce and control 
pollution, EPA has so far concentrated efforts in two areas: (1) monitoring 
technology, designed to determine the what, where, and how much of 
undesirable inputs into our environment, and (2) control technology and 
regulations designed to roll back the tide of effluents which threaten our health 
and the quality of our life. These efforts, of course, are appropriate and need 
to be continued without let-up, but they are essentially negative in approach 
since they indicate to industry and to people in general what they must not do, 
but not what they can do. I believe the time has come to add two positive 
dimensions to the menu; namely, (1) waste-management systems that couple 
in-house waste treatment with the assimilatory capacity of surrounding natural 
ecosystems that serve as the ultimate tertiary treatment plants, and (2) a 
merging of ecologic and economic assessments, along lines mentioned in my 
earlier review of theory so as to demonstrate what we all believe to be true; 
namely, that the economic return of clean environments is greater than the 
short-term gains that may result from ignoring or postponing pollution 
abatement. 


vi 


As an example of the first of these suggested new research areas, I can cite a 
project that we at the University of Georgia have undertaken under contract 
with a large industrial company. In this case the company proposes to build a 
chemical plant on a site that is adjacent to an extensive area of natural 
wetlands, both swamp forests and marshes, which we believe have considerable 
capacity to assimilate and recycle nutrients and bio-degradable wastes. By both 
inventory and experimental procedures we are in the process of determining 
just what this “tertiary” treatment capacity is with the understanding that the 
company will then design their in-house treatment facilities so as to remove the 
toxic substances and release into the wetland environment only that which can 
be assimilated. Such a procedure I like to call “reciprocal design” in that both 
the industrial engineer and the ecologist have the same objective; namely, 
essentially zero pollution after effluents have passed through both the 
man-made and the natural filters. In this case, the company owns the wetlands 
which, when used in the manner described, become a highly valued part of a 
total waste management system. I believe there would be much to be gained if 
EPA laboratories entered into “reciprocal design” contracts with industry, and 
thus become partners, rather than adversaries in the pursuit of common goals. 


Merging economics and ecology may prove difficult, but it does make 
common sense since the two words have a common Greek root, “oikos” 
meaning “household”; ecology literally is “the study of the household,” and 
economics “the management of the household.” The trouble is that “nature’s 
house” is entirely external to “man’s house” in current economic procedures, 
so that the very valuable and necessary work of nature, such as the tertiary 
treatment of wastes just discussed, is not included in economic cost-accounting 
or in the workings of the market system. In discussing theory, I made a point 
of the need to recouple the “houses” of man and nature, so we can follow up 
by suggesting that the best practical way to do this is to find ways to 
internalize into the economic system what are now considered to be the “free 
goods and services” of nature. 


I will close by mentioning several special marine research challenges, since 
this laboratory focuses on coastal and marine environments. Microbial 
components and transformations in marine and estuarine environments are the 
least known, yet the most important aspects when it comes to systems 
metabolism and the impact of man-made perturbations. Microbial activities in 
the anaerobic layers of sediments and how these activities are coupled with 
those in the aerobic layers and water columns provide especially difficult, but 
challenging, problems. The role of the sea in global cycles of carbon, nitrogen 
and sulfur need further study. For example, the sea has not proved to be as 
efficient a “sink” for CO ? released into the atmosphere by fuel-burning and 
deforestation, as had once been predicted. Finally, the impact of estuaries and 


Vll 


coastal wetlands on continental shelf waters needs close reexamination on local 
and regional scales. We now have a pretty good understanding of upwelling 
processes, but not of outwelling processes. On the basis of our early work at 
Sapelo, we thought that the salt marsh estuaries exported large quantities of 
detritus, but now we are not so sure if it’s POC, DOC, or living biomass that 
outwells, if indeed there is a net export at all. There is likely wide regional 
variation in import and export flows of carbon and nutrients along our 
coastline, and these need to be quantified if we are to anticipate the fate of 
pollutants which in the future are going to be introduced offshore (off-shore 
drilling, etc.) as well as inshore. 


TABLE OF CONTENTS 


TITLE PAGE 

Preparation and Characterization of a Marine Reference Material 1 

for Trace Element Determinations 

The Release of Heavy Metals from Reducing Marine Sediments 9 

The Use of Introduced Species ( Mytilus edulis ) as a Biological 26 

Indicator of Trace Metal Contamination in an Estuary 

Trace Metal Speciation and Toxicity in Phytoplankton Cultures 38 

A Simple Elution Technique for the Analysis of Copper in 62 

Neanthes arenaceodentata 

Geochemistry of Fossil Fuel Hydrocarbons in Marine Sediments: 68 

Selected Aspects 

Identification of Environmental Genetic Toxicants with Cultured 79 

Mammalian Cells 

Development of a Bioassay for Oils Using Brown Algae 101 

Effects of No. 2 Heating Oil on Filtration Rate of Blue Mussels, 112 

Mytilus edulis Linne 

Lobster Behavior and Chemoreception: Sublethal Effects of 122 

Number 2 Fuel Oil 

Influence of No. 2 Fuel Oil on Survival and Reproduction of 135 

Four Marine Invertebrates 

Extraction of Environmental Information Stored in Molluscan 157 

Shells: Application to Ecological Problems 

Laboratory Culture of Marine Fish Larvae and Their Role in 176 

Marine Environmental Research 

Laboratory Culture of the Grass Shrimp, Palaemonetes vulgaris 206 

Evaluation of Various Diets on the Lipid and Protein Composition 214 

of Early Life Stages of the Atlantic Silverside 


IX 


TABLE OF CONTENTS (Continued) 

TITLE PAGE 

The Combined Effect of Temperature and Delayed Initial Feeding 234 

on the Survival and Growth of Larval Striped Bass Morone 
saxatilis (Walbaum) 

The Evolution of the Bugsystem: Recent Progress in the Analysis 251 

of Bio-Behavioral Data 

The Effects of Temperature, Light, and Exposure to Sublethal 273 

Levels of Copper on the Swimming Behavior of Barnacle Nauplii 

Use of a Laboratory Predator-Prey Test as an Indicator of 290 

Sublethal Pollutant Stress 

Burrowing Activities and Sediment Impact of Nephtys incisa 302 

Second Generation Pesticides and Crab Development 320 

Statistical Linear Models in the Collection and Analysis of 337 

Ecological Data 

Kaneohe Bay: Nutrient Mass Balance, Sewage Diversion, and 344 

Ecosystem Responses 

Replicability of MERL Mocrocosms: Initial Observations 359 

Turbulent Mixing in Marine Microcosms — Some Relative Measures 382 

and Ecological Consequences 


x 


PREPARATION AND CHARACTERIZATION OF 
A MARINE REFERENCE MATERIAL FOR 
TRACE ELEMENT DETERMINATIONS 


Peter F. Rogerson and Walter B. Galloway 
Environmental Research Laboratory 
U.S. Environmental Protection Agency 
South Ferry Road 
Narragansett, R.l. 02882 


ABSTRACT 

A reference material for marine molluscan trace element determinations has 
been developed. It consists of 637 clams, Arctica islandica, that have been 
homogenized together and subsequently divided into 476 samples. A 
representative subsample of these has been analyzed for trace element 
concentrations. Of the 14 elements measured, 10 had relative standard 
deviations from the mean of 7% or less. 

INTRODUCTION 

The study of pollution in marine systems often involves the measurement of 
trace element concentrations in organisms over extended periods of time (1,2). 
Development of valid time trends from such data requires a strict quality 
control program at every stage of data collection, from field sampling through 
final statistics, to ensure that data from any single point in time is comparable 
to that collected at all other times. This paper describes some of the efforts 
undertaken to provide control over the laboratory analysis of marine organisms 
for trace element concentrations. Specifically, we describe the preparation and 
characterization of an in-house reference material which can be used as a 
benchmark sample for quality control, a known sample for methods 
development, or an intercalibration sample. 

EXPERIMENTAL 

Marine molluscan samples are prepared for flame atomic absorption 
spectroscopy as follows (3): 

1. Thaw sample. 

2. Using stainless steel instrument, shuck into a dry, labeled, tared 
beaker. Determine wet weight. 


1 


3. Cover beaker with watch glass and place in drying oven. Dry to 
constant weight at 95°C (about 48 hours). 

4. Cool and determine dry weight. 

5. Add concentrated nitric acid in sufficient quantity to cover 
organisms, adding acid in 20 ml increments. Cover beaker with 
watch glass. (Use same amount of acid for all samples within a 
group.) 

6. Let sample cold digest until tissue is well broken down; i.e., 4-24 
hours. 

7. Heat gently, to about 40°C, being careful to avoid frothing over. 
Continue until frothing stops. 

8. Heat to 85°C while covered, and bring sample to near dryness. (Be 
careful not to take to complete drymess at any time.) 

9. Remove from hot plate and add 20 ml of concentrated nitric acid. 
Repeat step 8. 

10. Repeat step 9 until digestion is complete, which is indicated by pale 
yellow color, clarification of the liquid, and no trace of lipids. 
(Treat all samples within a group in identical fashion.) 

11. Take to near dryness (about 5 ml remaining), cool, and add 20 ml 
of 5% nitric acid, getting all soluble residue into solution. 

12. Filter sample into 50 ml volumetric flask through Whatman #42 
Filter Paper which has been prerinsed with 5% nitric acid. 

13. Rinse beaker 2-3 times with 5% nitric acid and pour through filter. 

14. Rinse funnel down and bring up to volume with 5% nitric acid. 

15. Mix well, transfer to acid stripped 60 ml polyethylene bottle, and 
hold for A.A. analysis. 

NOTE: All glassware is detergent washed, soaked in 10% nitric acid, and 

copiously rinsed with deionized water. 

Each group of 1 5 samples is accompanied by a complete reagent methods 
blank. Analysis is performed on a Perkin-Elmer model 603 Atomic Absorption 


2 


spectrometer according to the manufacturer’s instructions. Operation is 
facilitated by the use of an autosampler (P.E. Auto 200) and an ASR-33 
teletypewriter. To ensure against instrument drift, a calibration standard is 
included with each 15 samples. To check for unknown matrix effects, a known 
spike is added to an aliquot of one of the samples and an equal quantity of 5% 
nitric acid. From this, a spike recovery is calculated for each group of 15. For 
each group, a sample of the reference material described in this paper is 
included. 

Preparation of Reference Material 

Clams (Arctica islandica) were collected by commercial dredge from Block 
Island Sound, and frozen prior to use. At the time of preparation, they were 
cleaned, thawed and shucked as if for analysis, except that extracellular fluid 
was drained and discarded. A total of 637 clam meats was pureed and 
homogenized in a stainless steel, 40 quart mixer, of the sort found in many 
commercial kitchens (Hobart VCM-40). Samples of approximately 60 g wet 
weight were removed, placed in 120 ml acid-stripped polyethylene bottles, 
sequentially numbered, and frozen for future analysis. This procedure yielded 
476 samples. 

Characterization of Reference Material 

From the 476 samples, a total of 65 were selected for investigation of the 
homogeneity of the material. These consisted of every tenth sample, and two 
blocks of 10 consecutive samples from each end of the sequence. These 
samples were analyzed for 14 trace elements by the above procedure, and the 
results examined for homogeneity. 

RESULTS AND DISCUSSION 

The concentrations of 14 metals in the reference material on a wet weight 
basis are listed in Table 1-1. Only data on a wet weight basis will be discussed 
because of some anomalous wet to dry weight ratios indicating some samples 
were not uniformly dried. On the basis of several criteria, the 14 metals may be 
divided into two groups, A and B. Group A consists of the 10 metals Cd, Cr, 
Cu, Fe, Mg, Mn, Ni, Pb, V, and Zn, for which the relative standard deviations 
are less than 7%, as seen in Table 1-2. Three of these metals are graphically 
represented in Figure 1-1. Note the similarity of the graphs. This similarity may 
be quantified for these metals by determining the 45 pair-wise correlation 
coefficients. Almost all of the coefficients indicate positive correlation at the 
95% confidence level, with many of them being much more highly significant. 


3 


Table 1-1. Trace Element Concentrations in Reference Material, ug/g (Wet) 



4 


Corrected for blank 









Table 1-1. (Continued) 



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Corrected for blank 












REFERENCE SAMPLE NUMBER 


Figure 1-1. Trace element distributions 
in reference material. 


6 












Table 1-2. Relative Standard Deviations 


Group A 

Metal 

Mean, ug/g Wet 

St. Dev. 

Rel. Std. Dev. 

Cd 

0.17 

0.01 

6 

Cr 

1.01 

0.05 

5 

Cu 

2.82 

0.07 

3 

Fe 

88.7 

4.5 

5 

Mg 

802 

58 

7 

Mn 

3.15 

0.10 

3 

Ni 

1.48 

0.06 

4 

Pb 

1.27 

0.04 

3 

V 

0.9 

(0.05) 

6 

Zn 

17.3 

0.5 

3 

Group B 

Al 

17.8 

2.5 

14 

Ca 

439 

61 

14 

Co 

0.19 

.04 

21 

Ti 

0.8 

0.3 

38 


In contrast are the group B metals which consist of Al, Ca, Co, and Ti. As 
shown in Table 1-2, these metals are characterized by a much higher relative 
standard deviation, from 14% to 38%. When plotted in a fashion similar to 
Figure 1-1, they show a much higher degree of scatter. Accordingly, the 
correlation coefficients show no real correlations at the 95% confidence level. 

Although this material has been prepared as a reference material, it is in no 
way equivalent to a standard reference material such as those developed by the 
National Bureau of Standards (NBS) (4). The NBS Standard Reference 
Materials are certified as to their trace element content on the basis of analysis 
by at least three independent techniques. Our material is an internal reference 
material, not a certified standard. 

This material is now in routine use within our laboratory. One sample, 
randomly chosen from the sequence, is included with each group of 15 
unknown samples prepared and analyzed. Thus, there is continuous verification 
of analytical results irrespective of operator, instrument settings, or standards. 
In addition, this material is ideal for methods development and intercalibration 
because of its extremely low variability. Individual organisms from a wild 


7 







population can have a range of metal concentrations up to an order of 
magnitude, thus obscuring differences between techniques that may be small 
but real. Comparison of clam homogenate values, however, will render obvious 
differences of less than 25% which would be arduous or impossible to discern 
using natural populations. 

CONCLUSIONS 

We have shown that a satisfactorily homogeneous reference material can be 
prepared with a minimum of specialized equipment. The material has a low 
relative standard deviation for the 10 metals Cd, Cr, Cu, Fe, Mg, Mn, Ni, Pb, V, 
and Zn. For reasons that are not always clear, the other metals analyzed Al, Ca, 
Co, and Ti have higher relative standard deviations. The material is useful as a 
benchmark reference material so that analyses at different times can be shown 
to be intercomparable. Thus, legal defensibility of time series studies of 
pollution sites can be greatly enhanced. The material is also ideal for methods 
development and intercalibrations because of its very low relative standard 
deviation. Therefore, with cooperation from a cafeteria kitchen, it becomes a 
relatively simple task to prepare such a reference material when a certified 
standard from a recognized supplier such as NBS is not available. 

ACKNOWLEDGEMENTS 

We gratefully acknowledge the statistical consulting of Drs. J. Callahan and 
J. Heltshe of the University of Rhode Island, and the assistance of Dr. R. Payne 
and Messrs. B. Reynolds, D. D’Alessio, F. Storti, E. Truesdell, and C. Young of 
this lab. 

REFERENCES 


1. Eisler, R. 1973. Annotated Bibliography on Biological Effects of Metals in 
Aquatic Environments, 287 pp. Washington, D.C.: U.S. Government Printing 
Office (U.S. Environmental Protection Agency, Rep. No. R3-73-007). 

2. Eisler, R. and M. Wapner. 1975. Second Annotated Bibliography on 
Biological Effects of Metals in Aquatic Environments, 400 pp. Springfield, 
Virginia: National Technical Information Service. (U.S. Environmental 
Protection Agency Rep. No. 600/3-75-008). 

3. Pesch, G., B. Reynolds, and P. Rogerson. 1977. Trace Metals in Scallops 
from Within and Around Two Ocean Disposal Sites. Mar. Pollut. Bull. <5 p. 
224. 

4. Anonymous. 1975. Catalog of NBS Standard Reference Materials. NBS 
Special Publication 260. p. 32. 


8 


THE RELEASE OF HEAVY METALS 
FROM REDUCING MARINE SEDIMENTS 

Michael L. Bender 
Richard J. McCaffrey 
J. Douglas Cullen 
Graduate School of Oceanography 
University of Rhode Island 
Kingston, Rl 02881 


ABSTRACT 

We address the hypothesis that metals forming soluble sulfides are released 
from nearshore anoxic sediments, while those forming insoluble sulfides are 
retained. As a test, we have studied pore water chemistry, benthic fluxes, and 
water column distributions of heavy metals in Narragansett Bay, Rhode Island. 
The results show that metal forming soluble sulfides (Mn and Fe) have high 
pore water concentrations and are released to the Bay waters, while metals 
forming insoluble sulfides have low pore water concentrations and negligible 
benthic fluxes. 

INTRODUCTION 

Several authors have suggested that heavy metal concentrations in reducing 
marine waters are strongly influenced by sulfide solubility: metals forming 
relatively soluble sulfides (such as Fe and Mn) will have relatively high 
concentrations, and those forming relatively insoluble sulfides (such as Cd and 
Cu) will have very low concentrations (5, 13, 4). A corollary of this suggestion 
is that benthic fluxes (fluxes of dissolved chemicals from sediments to the 
overlying water) out of anoxic sediments should be high for those metals 
forming soluble sulfides, and negligible or even negative for those metals 
forming insoluble sulfides. These hypotheses, if correct, have important 
implications. They imply that estuarine and reducing nearshore and continental 
shelf sediments are not sites where insoluble sulfide-forming metals are readily 
re mobilized: metals reaching these sites by inorganic scavenging or biological 
removal are, most likely, permanently sequestered as long as sulfide is being 
produced in the sediment porewaters. Secondly, the hypotheses indicate that, 
to avoid release of metals forming insoluble sulfides, sewage sludge and dredge 
spoils polluted with heavy metals should be dumped under sites of high organic 
productivity, where continued deposition of organic matter maintains the 
conditions which prevent their release to the overlying water. Of course, other 
considerations (such as the presence of organic toxins) may show another 
course of action to be more prudent. 


9 


We have carried out a series of experiments aimed in large part at testing the 
two hypotheses outlined above. We have proceeded by 1) determining the 
concentrations of elements forming relatively soluble sulfides (Mn and Fe) and 
elements forming relatively insoluble sulfides (Ni, Cu and Cd), along with 
nutrients, pH, total CO-, (TCO->), SO^ - , and S in anoxic Narragansett Bay 
sediments; 2) carrying out tracer experiments to determine the rate at which 
organisms enhance the benthic flux of chemicals by pumping water across the 
sediment-water interface, and using these results, along with diflusive flux 
estimates, to predict total fluxes across the sediment-water interface; 
3) measuring benthic fluxes directly via bell jar experiments, and using the 
results both to check the prediction of the pore water data and flux model, and 
to make direct determinations of metal benthic fluxes; and 4) working out the 
mass balance of heavy metals in Narragansett Bay at one point in time, using 
the Bay itself as a gigantic bell jar and thereby checking on the generality of 
our benthic flux measurements. 

In this paper we summarize the results of this work. 

PORE WATER WORK 

Samples were collected in plexiglas box cores or PVC pipe. One centimeter 
sediment slices were placed in polyethylene centrifuge bottles in a helium 
atmosphere and centrifuged at in situ temperatures to separate pore waters. 
Supernatant waters were filtered through acid-washed Nuclepore filters. The 
concentration of TCO-, was determined by gas chromatography. Sulfate was 
measured by BaSo^ coprecipitation using ^ ^Ba as a tracer. Sulfide and 
ammonia were determined colorimetrically by the methods of Cline (3) and 
Solorzano (10), respectively. Phosphate and silicate were determined by a 
modified autoanalyzer procedure (11, 12). Dissolved manganese and iron were 
determined by flameless atomic absorption spectrometry using either a 
Perkin-Elmer 503 or 360 AAS with model 2100 graphite furnace deuterium arc 
background corrector. Standards were prepared in Sargasso Sea water. Further 
details of sample collection and the above analytical methods are given by 
McCaffrey (8). 

Cd and Ni were determined by flameless atomic absorption after sample 
concentration using a modification of the Co-APDC coprecipitation technique 
of Boyle and Edmond (2). A 0.2 ml aliquot of a 4mM CoCl-> solution is added 
to the pore water sample at pH 2, followed by addition of 0.4 ml of a 2% W/V 
solution of APDC. The sample is shaken vigorously and allowed to sit for up to 
30 minutes. It is then filtered through a 2.5 cm diameter, 0.4 jum micron 
Nuclepore filter held by an acid-washed Millipore Filter apparatus. The filter is 
washed with several ml of deionized water, and placed in a polypropylene vial 
containing 1.0 ml of redistilled 3N HNO^. The precipitate is dissolved by 


10 


ultrasonication tor 1 hour. The filter is then removed and the solution analyzed 
by fiameless atomic absorption. 


The yields, as determined by addition for Ni and Cu and tracer experiments 
for Cd, are 0.76±.06%, 0.75±.10, 0.70±.08 for Ni, Cu and Cd, respectively. The 
piecision is about ± 10 % at concentrations well above the detection limits of 
0.1 ppb for Cd and 1.0 ppb for Cu and Ni. 

Equipment and reagents are all carefully cleaned prior to Ni, Cu and Cd 
analysis. Polypropylene vials are soaked in 6 M HC1 for 18 hours, rinsed with 
deionized water, and ultrasonicated twice for three hours each time in 6 M HC1. 
They are rinsed at least three times and dried. CoCl 2 6H 0 0 is purified^using 
Dowex-l-X 8 anion exchange resin. It is put on in 9M HC1 and eluted in 4M 
HC1. The APDC solution is purified by filtration through a 0.4 jum micron 
Nuclepore filter followed by five extractions with 20 ml MIBK (methyl 
isobutyl ketone). Filters are acid washed, as all glassware used in the filtration 
is continuously soaked in acid. Blanks are below the detection limits. 

Typical summer pore water profiles are shown in Figures 2-1 and 2-2 for a 
long core and four short cores from the Jamestown North study site in 
Narragansett Bay (located about 0.5 km north of Jamestown Island in 5-10 m 
of water). These profiles are discussed in detail by McCaffrey et al ( 8 ). 
Concentrations of all constituents are far higher in the top centimeter than in 
bottom water (TC0 2 increases from 2.0 to 2.8 mM, NH 3 from ~5 to 100 juM, 
PO 4 ~ from 1-25 juM, etc.). From 1-20 cm the profiles are flat (TCCh, NH 3 , 
HqSiOq) or show decreasing concentrations with depth (PO 4 = , Mn ++ ). The 
lack of a systematic increase is ascribed to transport of metabolites out of the 
sediments by the pumping activity of organisms, rather than by ionic or 
molecular diffusion. The sharp concentration decreases observed for Mn ++ and 
PO 4 = are ascribed to inorganic reaction in the sediment column. Below 
approximately 25 cm, SO 4 = concentrations decrease and concentrations of 
other metabolites increase. Organisms are assumed to be absent, or at least 
ineffective water transporters, below this depth. Metabolite concentrations 
change sympathetically approximately as predicted by organic matter 
decomposition: TC0 2 increases twice as fast as SO 4 — decreases NH 3 increases 
about 1/7 as rapidly as TCO?, and PO 4 = increases about 1/150 as rapidly as 

tco 2 . 

An important point about the flat portion of the profiles is that the TC0 0 
value is considerably higher than can be accounted for by 0 -> reduction, and 
implies anoxic diagenesis. The bottom water TCO-> and 0 o concentrations are 
2.0 and 0.15 mM, respectively. When all 0 2 is consumed, the TC0 0 
concentration will rise to 2.15 mM. NO 3 reduction could conceivably increase 
the value to 2.2 mM. SO 4 - reduction must be postulated as the agent causing 
the further increase to 2.8 mM. 


11 


ZC0 2 (mM) SO/ or S = (mM) NHj(mM) PO/ (/xM) SiO 


o 


O o o 


o 


o o 




(UJO) Hid 3 a 


12 


Figure 2-1. Concentrations of TC0 2 , SC> 4 _ , S _ , NH 3 , P 04 - , and H4SiC>4 in pore waters 
from a Jamestown North long core (JN-8) (from McCaffrey et al, 1977). 












SC0 2 (mM) N H 3 (^xM) P0 4 = (/xM) Si0 2 (/xM) Mn (ppb) 

3 0 100 200 0 50 100 0 200 400 600 0 1000 2000 









13 


Figure 2-2. Concentrations of TCC> 2 , NH 3 , PO 4 -, H 4 Si 04 and Mn ++ vs. depth in pore waters 

of the long core (NJ- 8 ) and four short cores from the Jamestown North study site. 

Note: Heavy lines show inferred concentration gradients at the sediment-water 
interface (from McCaffrey et al, 1977). 










































Nutrient and metal data for Jamestown North core 11 (a short core 
collected on 7/13/76) and 12 (a long core collected on the same date) are given 
in Tables 2-1 and 2-2. Metabolite concentrations vary in the manner discussed 
earlier. 

The metal concentrations are striking. Mn and Fe values are similar to values 
reported elsewhere for anoxic sediments (11). Cu and Cd concentrations, on 
the other hand, are very low, being comparable to or less than Bay bottom 
values of about 2 ppb (Cu) and 0.2 ppb (Cd) at the Jamestown North location. 
There is some scatter in the Cu and Cd data. This is in part due to 
contamination. For example, the 58-60 cm sample in Jn-12 has Mn, Fe and Cd 
values which are all higher than values in surrounding samples, apparently due 
to contamination. On the other hand, there may be real variations, such as a Cu 
maximum at 3-6 cm in JN11. Nevertheless, the main conclusion is clear: of the 
four metals we analyzed, those forming relatively soluble sulfide (Mn and Fe) 
gave pore water concentration far above ambient bottom waters, whereas those 
forming highly insoluble sulfides (Cu and Cd) gave concentrations comparable 
to or less than bottom water values. We would not expect such sediments to 
release Cu and Cd to the bottom waters at significant rates. 

MODELLING SEDIMENT-WATER EXCHANGE 

To understand how fluxes of constituents between pore and overlying 
waters at Jamestown North depend on pore water and bottom water 
distributions, McCaffrey et al (8) constructed a simple model in which we 
consider both exchange mechanisms - simple diffusion (transport along 
gradients due to the thermal motion of ions and molecules) and advection 
(transport in water which is moving as a result of the irrigation, feeding and 
burrowing activities of the benthic fauna). 

Conceptually, the diffusive flux is easy to calculate - it is simply the 
product of a diffusion coefficient and a concentration gradient. Diffusion 
coefficients in Narragansett Bay sediments at 25°C were taken as half the value 
at 25 C in deionized water. Concentration gradients are not well known: the 
gradients are very steep in the top 1 centimeter and zero below, and the pore 
water concentrations do not allow us to accurately estimate the gradients at 
the interface. We have assumed that the concentration near the interface is as 
shown in Figure 2-2. Diffusive fluxes were calculated from the product of the 
gradient and the diffusion coefficient. 

Allei (1) and others have stressed the importance of organisms in 
sediment-water exchange. In calculating advective fluxes, it is necessary to 
know the rate at which organisms move water. The activity of organisms may 
either be modelled as a random, or “biodiffusion” process, or as an ordered, or 
“biopumping” process. Since organisms pump water into the sediment'to 


14 


Table 2-1. Nutrient and Metal Concentrations in Pore Waters from a Short Core 

from the Jamestown North Study Site (JN-11) 



.—. 














-Q 

Q. 

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cm 


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cm 

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3 

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1 

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Csl 

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15 


11- 12 2.75 26.3 <0.003 57 12.1 160 85 .16 1.4 4.1 

12- 13 2.35 26.5 <0.003 51 11.1 155 42 .16 <1 2.6 

13- 14 2.50 26.2 <0.003 45 10.1 148 44 <.10 8.4 13.4 

14- 15 2.50 24.3 0.003 41 8.7 122 101 -10 <1 2.6 




Table 2-2. Nutrient and Metal Concentrations in Pore Waters from a Long Core 

from the Jamestown North Study Site (JN-12) 


D 

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Q. 

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S -I 


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16 








supply oxygen and pump water out to expel waste products, we regarded the 
biopumping model as more appropriate. In the model of McCaffrey at al (8), it 
was assumed that organisms pumped water across the sediment-water interface 
at a certain “biopumping rate” (with units of volume of water per unit surface 
area per unit time). The biopumping rate was determined experimentally by 
bringing box cores of Jamestown North sediment into the laboratory, spiking 
the supernatant solution with ^^Na, and measuring the decrease in the 
supernatant “~Na concentration with time. The experiments gave a 
biopumping rate of 0.7±0.3 cm^ cm'“ day'^. The biopumping flux is then 
taken as the product of the biopumping rate and the difference between pore 
water and bottom water concentrations. 

Model diffusive and advective fluxes for summer Jamestown North 
sediments are given in Table 2-3. Surprisingly, both fluxes are of the same 
magnitude. 

RESULTS OF BENTHIC FLUX MEASUREMENTS 

In order to test our predictions that nutrient and manganese fluxes have the 
values calculated from the model outlined in the previous section, and to make 
direct measurements of copper and nickel fluxes, we have measured benthic 
fluxes in the field using the “bell jar” instruments developed and extensively 
deployed by Hale (7) and Nixon et al (9). In these experiments, PVC pipe 
halves with closed ends PVC flanges around the base are placed on the 
sediment. At the start of the experiment, a sample is withdrawn from the 


Table 2-3. 



Diffusive 

Flux 

Advective flux 

calculated from 
biopumping model* 

Measured 

fluxes: 
x ± 1 <7 (n) 

H^SiO^ 

0.3 

0.3 

1.2±0.2(7) 

nh 3 

0.19 

0.07 

0.27±0.08(15) 

III 

o 

CL 

0.018 

0.02 

0.07±0.02(9) 

£C0 2 

0.9 

0.6 

2 ( 1 ) 

.. ++ 

Mn 

0.02 

0.01 

0.049±0.018(14) 


3 -2 -1 

* Assuming a biopumping rate of 0.7 cm cm day 


17 




chamber from a valve at one end; a second sample is withdrawn at the end of 
the experiment. An ambient bottom water sample is also taken, and a dark 
bottle is filled with water at the start of the experiment and sampled at the 
end. The samples are analyzed for metals and nutrients after filtration. Benthic 
fluxes are calculated from the change in the concentration of a constituent in a 
chamber, the mean height of the chamber (chamber volume/enclosed sediment 
surface area), and the length of the experiment. Dark bottle “fluxes” are 
calculated in the same manner as benthic fluxes, taking the dark bottle 
concentration at the end of the experiment as the final concentration, and the 
ambient bottom water concentration as the initial concentration. The dark 
bottle results are important for metals; because if they are equal to zero within 
analytical uncertainties, they indicate that metal analyses are not seriously 
affected by sample contamination during collection. 

Metal concentrations were measured with the techniques used for pore 
waters. The ambient water column metal concentrations at Jamestown North 
are about 10 ppb (0.2 juM) for Mn and Fe, 3 ppb (0.06 nM) for Ni, 2 ppb (0.04 
juM) for Cu, and 0.1 ppb (.001 juM) for Cd. Precisions in individual metal 
analyses are ±10%; hence uncertainties in fluxes are 40 juM m'“ day'^ for Mn 
and Fe, 8 juM m‘- day*^ for Cu and Ni, and 2 /iM m'~ day’* for Cd. Flistograms 
of dark bottle results at Jamestown (Figure 2-3) are roughly as predicted from 
these errors. 

Results of experimentally determined fluxes of Mn ++ and nutrients at 
Jamestown North are given in Table 2-3, along with model fluxes for these 
chemicals presented earlier. The model fluxes agree quite well with the 
measured values, indicating that the system is well characterized. 

A histogram of Jamestown North metal fluxes is shown in Figure 2-4 , and 
averages are tabulated in Table 2-4. Manganese fluxes are similar to the model 
values. Pore water iron concentrations are similar to those of manganese, and 
comparable fluxes are predicted. Observed iron fluxes are about an order of 
magnitude lower than manganese (and predicted) fluxes; this is ascribed to 
rapid oxidation of iron in the supernate following diffusion out of sediments. 
Nickel, copper, and cadmium fluxes are predicted to be negligible (see 
discussion of pore water values, above) and in fact measured fluxes are 
generally equal to zero within the analytical uncertainty. 

From the concentration of constituents in the Bay, the average height of the 
water column (taken as 10 m) and the fluxes, we can calculate doubling times 
of metals in the Bay with respect to benthic fluxes. These values are given in 
Table 2-4. Upper limits on Cu, Ni and Cd doubling times were calculated taking 
the flux as less than or equal to the sum of the mean flux and one standard 
deviation. Doubling times are to be compared with residence times of water in 
Narragansett Bay of about one month. The results show that, in the 


18 


DARK BOTTLE RESULTS AT 
JAMESTOWN ( M nrf 2 day -1 ) 

N 




liJ I i i i i I i i 

<-500 -500 -250 


j ii 


♦i i i i i i*i i i i 


250 500 



Ni 


• • 


i i i i i i i i»i«i 


•l«l I I I I I I I I LI 


-50 -25 


Cu 


0 

(I) 


25 50 >50 


• • 


I I I l l I I I l»l» 


l l*M»l I l I I I 


-50 -25 0 


Cd 


25 50 


• • 

I I M !•! I I 1 * 1 * 


*1*1 1*1 1 I I 1 I J 


- 2.0 - 1.0 


1.0 2.0 


Figure 2-3. Histograms of dark bottle results at Jamestown North, 
calculated as fluxes (see text). Units are mM m‘2 day’^. 


19 












TRACE METAL FLUXES AT 
JAMESTOWN (/iM m' 2 day"') 

N 


t*J 

<-500 



I^J 


-500 -250 


0 250 500 >500 

+ FLUX —► 



[•) 


-1000 -500 


0 


500 1000 >1000 



I 1 I 1*1 M l*l*l 


1 1 1*1 1 I 1 1 


-50 -25 


0 

(I) 


25 


Jj 

50 




Cu : 

• 

• 

• 

• 

• 

• • 

111M11 

_• • 

• • 

~»i»i 11111111 


-50 -25 0 25 50 

( 2 ) 


Figure 2-4. 


Cd 


I 1 1*1 I l*l*l*l*l 
2.0 - 1.0 


1 *U 1 


0 


1 J 1 1 i I 
1.0 2.0 


Histograms of heavy metal fluxes at Jamestown North 
(units are /uM nr 2 day* 1 ). 

20 























Table 2-4. Benthic Fluxes Measures at the Jamestown 
North Study Site and Estimate Doubling Times 
for Cu, Ni, Mn and Fe in Narragansett Bay 


Mean flux and Concentration of Time for benthic flux 

standard deviation dissolved metal in to double water column 

(jug cm' 2 day* 1 (1) Narragansett Bay (ppb) concentration (days) 


Cd 
Cu 
Ni 
Mn 
Fe 

NOTES: 

(1) Based on twelve determinations. 

(2) Calculated excluding one anomalously high value believed to reflect 
contamination. 


-0.0029±0.0043 

0.10 

>71 

-0.009±0.044 

2.0 

>57 

-0.035±0.064 

3.0 

>100 

2 .1±0.8 (2) 

10 

5 

0.17±0.23 




summertime, release of manganese from sediments is a major source of 
manganese in Narragansett Bay, but release of nickel, copper and cadmium are 
probably not significant. 

TRACE METAL BUDGETS IN NARRAGANSETT BAY: 

Mn AND Cu AS EXAMPLES 

The preceeding discussion suggests that benthic fluxes will be a source of 
Mn, but not Cu, to the waters of Narragansett Bay. The distribution of these 
metals in the Bay provides a check on these conclusions, as will be seen from 
the following discussion. 

Graham et al (6) did a mass balance for dissolved and particulate manganese 
in Narragansett Bay in the Summer of 1973. Their results for the distribution 
of dissolved manganese in the main part of the Bay are shown in Figure 2-5. It 
is readily seen that dissolved manganese is not a single-valued function of 
salinity: at a given salinity, surface waters have far lower manganese 
concentrations than deep waters. The manganese concentration of surface 
samples is far less than that expected from a simple conservative mixing model 
in which the deep water concentration is approximately equal to the 
conservative concentration. Graham et al (6) interpreted these results as 
indicating that manganese is scavenged (presumably by oxidation and 
precipitation) throughout the waters of the estuary, and bottom waters are 
enriched relative to surface waters by the benthic flux of manganese. 


21 








SALINITY, %o 

Figure 2*5. Dissolved Mn vs. salinity in Narragansett Bay, 

in the Summer of 1973. 

Dissolved copper and manganese (along with 0*,, nutrients and other metals) 
were measured in the waters of Narragansett Bay in the Spring of 1977 by the 
Narragansett Bay Study Group (in preparation). At this time of year, 
manganese was found to be nearly conservative, showing no surface water 
depletion and only a small deep water enrichment. The contrast between 
manganese behavior in the spring and summer probably reflects slower 
oxidative precipitation, slower benthic fluxes, and more rapid flushing of the 
Bay under springtime conditions of lower temperatures and higher runoff. 


22 





The dissolved copper distribution in Narragansett Bay in the springtime of 
1977 is shown in Figure 2-6 (results of the Narragansett Bay Study Group). 
Concentrations were determined using the methods outlined earlier for pore 
waters. Several copper values fall far above a conservative mixing line, and are 
believed to reflect contamination. Most values, however, appear to define a 
simple conservative mixing line. These results show no evidence for an input of 
copper into the Bay by diffusion out of sediments. The Bay water results will 
be discussed in more detail elsewhere. 


_o 

CL 

CL 


cr 

LU 

CL 

CL 

o 

o 

o 

LJ 

> 

-J 

o 

CO 

CO 

o 


7 


6 


5 


4 


3 


2 


1 

24 26 28 30 32 34 

SALINITY, % 0 


t 


18 


NISKIN BOTTLE, SURFACE O 
( 1 m depth) 

NISKIN BOTTLE, BOTTOM □ 
PLASTIC BUCKET A 


□ A 


O ° 

A O 
O 


ZD 

u 


□ 


□ u □ 


A 

-A-L 


□ 


A 

-A 


Figure 2-6. Dissolved Cu vs. salinity in Narragansett Bay, 
in the Spring of 1977 (Narragansett Bay Study Group, in prep.). 


23 










SUMMARY 


The results summarized here are consistent with the hypotheses of earlier 
workers that metals forming highly insoluble sulfides will be sequestered in 
anoxic marine sediments. 

Our conclusions reflect results of a limited study on a small number of 
metals during one or two seasons in a single estuary. The conclusions are thus 
preliminary, and cannot be extrapolated to other seasons, metals or estuaries. 
Organic complexing, in particular, may render certain heavy metals far more 
soluble than would be expected from sulfide solubilities calculated, considering 
inorganic ion pairing only. 

ACKNOWLEDGMENTS 

We are grateful to Scott Nixon, Candace Oviatt and colleagues, for their 
generous cooperation in collecting samples, and for the loan of sampling 
equipment. We also wish to express our appreciation to Nile Luedtke, who 
participated extensively in the pore water and benthic flux determinations, and 
scuba divers Paul Benoit, George Morrison, Allen Myers and Bob Pavia for their 
skill and care in obtaining in situ benthic flux samples. This work was 
supported by a grant from the Environmental Research Laboratory of the 
Environmental Protection Agency. 

REFERENCES 

1. Aller, Robert C. 1977. The Influence of Macrobenthos on Chemical 
Diagensis of Marine Sediments. Ph.D. Thesis, Yale University, 600 pp. 

2. Boyle, Edward G. and John M. Edmond. 1975. Determination of Trace 
Metals in Aqueous Solution by APDC Chelate Coprecipitation. In: 
Advances in Chemistry Series, No. 147, Analytical Methods in 
Oceanography (Thomas R.P. Gibb, Jr., ed.) American Chemical Society, 
pp. 44-55. 

3. Cline, J.D. 1969. Spectrophotometric Determination of Hydrogen Sulfide 
in Natural Waters. Limnology and Oceanography 74:454-458. 

4. Elderfield, H. and A. Hepworth. 1975. Diagenesis, Metals and Pollution in 
Estuaries. Marine Pollution Bulletin 6:85-87. 

5. Goldberg, E.D., W.S. Broecker, M.G. Gross and K.K. Turekian. 1971. 
Marine Chemistry. In. Radioactivity in the Marine Environment, National 
Academy of Sciences, pp. 137-146. 


24 


6 . Graham, W.F., M.L. Bender and G.P. Klinkhammer. 1976. Manganese in 
Narragansett Bay. Limnology and Oceanography 21 :665-673. 

7. Hale, Staphen. 1974. The Role of Benthic Communities in the Nutrient 
Cycles of Narragansett Bay. M.S. Thesis, University of Rhode Island, 129 

pp. 


8 . McCaffrey, Richard J., Allen C. Myers, Earl Davey, George Morrison, 
Michael Bender, Nile Luedtke, Douglas Cullen, Philip Froelich and Gary 
Klinkhammer 1977. Benthic Fluxes of Nutrients and Manganese in 
Narragansett Bay, Rhode Island. Submitted to Limnology and 
Oceanography. 

9. Nixon, Scott W., C.A. Oviatt and S.S. Hale 1976. Nitrogen Regeneration 
and the Metabolism of Coastal Marine Bottom Communities. In: The Role 
of Terrestrial and Aquatic Organisms in Decomposition Processes (J.M. 
Anderson and A. Macfayden, eds.) Blackwell Scientific Publication, 
Oxford, pp. 269-283. 

10. Solorzano, L. 1969. Determination of Ammonia in Natural Waters by the 
Phenol-Hypochlorite Method. Limnology and Oceanography 74:799-801. 

11. Technicon 1973. Technicon Industrial Method 155-71/W. Orthophosphate 
in Water and Seawater. Technicon Industrial Systems, Tarrytown, N.Y. 
10591. 

12. Technicon 1973. Technicon Industrial Method 186-72/W. Silicate in Water 
and Seawater. Technicon Industrial Systems, Tarrytown, N.Y. 10591. 

13. Thompson, John, Karl K. Turekian and Richard J. McCaffrey 1975. The 
Accumulation in and Release from the Sediments of Long Island Sound. 
In: Estuarine Research, Vol. I, Chemistry, Biology and the Estuarine 
System, Academic Press, New York, pp. 28-44. 


25 






THE USE OF INTRODUCED SPECIES 
(MYTILUS EDULIS) 

AS A BIOLOGICAL INDICATOR OF TRACE 
METAL CONTAMINATION 
IN AN ESTUARY 


D. K. Phelps and W. Galloway 
EPA, Environmental Research Laboratory 
Narragansett, R.l. 02882 


The use of introduced as well as indigenous marine species as biological 
monitors or indicators of water quality is being evaluated at the Environmental 
Research Laboratory, Narragansett (ERLN). This paper presents data that 
demonstrate the edible blue mussel, Mytilus edulis, to be an effective indicator 
of metal pollution when introduced along a gradient of anthropogenic stress. 
M. edulis were collected from a commercially-fished mussel bed in Narragansett 
Bay, Rhode Island, and held in a laboratory seawater system for six days. 
Sub-groups were deployed in polluted and clean sections of that estuary, 
respectively, for a period of four weeks. 

Atomic absorption analyses revealed that M. edulis from the polluted section 
had significantly higher levels of lead, nickel, and copper when compared to 
Mytilus from the clean part of the estuary and those retained in the seawater 
system at the laboratory as controls. No differences were apparent between the 
three groups in the case of cadmium, chromium, vanadium, and zinc; however, 
comparisons between introduced Mytilus and indigenous Mercenaria 
mercenaria, demonstrated Mytilus to be an effective surrogate biological 
monitor for M. mercenaria in the case of lead, nickel, and copper. 

INTRODUCTION 

The area of study is Narragansett Bay, Rhode Island, U.S.A. (Figure 3-1). 
The Bay has been described as “abnormally stressed” by man’s activities in its 
upper reaches, and as being divisible into a polluted upper Bay, a transitional 
zone, and a lower Bay having water of high quality (1). Bottom water salinities 
range 28-31°/oo in the upper reaches, and 30-32°/oo in the lower Bay. 

Temperature seasonally escalates from freezing to 26^C in various sections of 
the estuary. 


26 



NARRAGANSETT BAY 

RHODE ISLAND 


Figure 3-1. Area of study. 


27 










Major environmental differences within the system are attributable to a 
history of pollution effects in the upper reaches of the Bay (Figure 3-1). Major 
sources of pollution are domestic waste treatment plants, which include 
industrial effluents from such activities as metal plating and jewelry 
manufacturing and urban runoff. 

Fine sediments in the upper area (Stations 1 and 2) are anaerobic, 
characteristically having the redox boundary at the sediment-water interface, as 
well as having a strong odor of H->S. In the lower Bay (Stations 3 and 4), fine 
sediments have a well-defined aerobic layer with a redox boundary defined 
between 5 and 10 cm below the sediment-water interface (2). Metals in upper 
Bay sediments include typically elevated levels of zinc (337 ppm), lead (167 
ppm), copper (493 ppm), and chromium (208 ppm) compared to lower Bay 
levels of zinc (119 ppm), lead (40 ppm), copper (48 ppm) and chromium (53 
ppm) (2). In addition, higher concentrations of hydrocarbons have been 
reported in upper Bay sediments compared to levels found in the lower Bay 
(3). Over the past few years, a transect of stations, indicated in Figure 3-1 as 1, 
2, 3, and 4, has been used to study the effects of pollution from north to south 
in the Bay. 

Mercanaria mercenaria is a molluscan species indigenous to all areas of the 
Bay. Phelps and Myers (5) compared levels of aluminum (Al), cadmium (Cd), 
cobalt (Co), copper (Cu), lead (Pb), manganese (Mn), nickel (Ni), silver (Ag), 
titanium (Ti), vanadium (V), and zinc (Zn) between Mercenaria collected from 
the “polluted’ upper Bay and the “clean” lower Bay. Of particular interest is 
the tact that alter a thirty-day period of depuration, Mercenaria from the 
upper polluted part of the Bay retained significantly higher levels of Cd, Cu, 
Ni, Pb, and Ti compared to Mercenaria collected from the lower “clean” part 
of the Bay (Figure 3-2). 


This paper reports on the use ot Mytilus ednlis as an introduced biological 
monitor for trace metals. The specific goals of the study were: 

1. To observe whether or not introduced Mytilus bioaccumulation would 
reflect the spatial differences in trace metal levels previously observed 
in sediments and indigenous Mercenaria. 

2. It such differences were reflected by Mytilus , over what time frame 
were differences observable. 

3. If quantitative difterences were reflected, how do metals accumulated 

by introduced Mytilus , compare qualitatively to metals accumulated by 
indigenous Mercenaria. 


28 


1000 — 


h- 

x 

O 

LU 


>- 

or 

Q 


CL 

CL 



Cu Ni Pb 

MERCENARIA 


Figure 3-2. Metal levels in indigenous Mercenaria mercenaria from 
clean and polluted parts of Narragansett Bay, Rl. 

Note: Range shown is one standard deviation on either side of the mean. 


The mussels used in this study were collected at one time from off 
Popasquash Point, Narraganasett Bay, R.I. (Figure 3-1), and held in a 
laboratory flow-through system for six days prior to the First deployment. 


29 




















































Field Methods 


Since metals were chosen as the initial pollutants of interest, a completely 
metal-free apparatus was desired for deploying mussels in the Field. In view of 
potential use by other investigators, a simple, relatively inexpensive apparatus 
which was easy to deploy and service was a secondary goal of the design 
process. The present apparatus is shown in Figure 3-3. 

A plastic float is attached to a concrete weight by approximately two meters 
of polypropylene line. The mussel holding baskets, 15 x 15 x 15 cm 
polypropylene test tube baskets with snap-on lids, are suspended from the line 
approximately 1 meter above the sediment surface. 

A Mytilus monitoring station is deployed in a two-step operation. The 
apparatus, minus the mussel baskets, is lowered to the bottom in 5 to 8 m of 
water, after which typically eight mussel baskets are taken down and 
suspended in groups of four from the line by SCUBA DIVERS. The 
deployment operation requires 10 to 15 minutes on-station and subsequent 
sampling of about five minutes. Each basket of 20 mussels serves as a 
subsample, thereby allowing removal of the desired sample without disturbing 
the remaining mussels. Samples for metals analyses are immediately transferred 
to “ziploc” polypropylene bags and frozen until analyzed. 


plastic 

float 

polypropylene 

line 

mussel 

baskets 





Approx. I m 


Figure 3-3. Schematic of subsurface mussel station. 


30 


















Current Study 


The first collection was made from each station after an exposure period of 
three weeks. A second collection was made one week later. 

Analytical Methods 

The mussel tissue is analyzed for metal content by flame Atomic Absorption 
Spectrometry after wet digestion in concentrated nitric acid. Each sample is 
oven dried to constant weight, then digested in concentrated nitric acid in a 
simple reflux system. The digestate is filtered on transfer to 50 ml volumetric 
flasks, brought up to volume, and analyzed on a Perkin-Elmer Model 603 
Atomic Absorption Spectrophotometer using Deuterium arc background 
correction where necessary. The raw data are reduced to ug of metal/gram of 
tissue, on both a set and dry basis, by computer. 

RESULTS 

Results of analyses for Cd, Pb, Ni, Cu, chromium (Cr), V, and Zn on Mytilus 
from Stations 2 and 3, as well as from controls in the Wet Lab, are presented in 
Figures 3-4 and 3-5 and Table 3-1. 

Except in the case of Pb and V, where the numbers of samples having 
detectable limits were below the minimum number required for the statistic, 
the standard deviation is comparable between a sample of 41 or 10 (Table 3-1). 
This fact establishes that 10 Mytilus are a reasonable sample size with the 
noted exceptions of Pb and V. After four weeks, Cd levels in Mytilus from 
mid-Bay, Station 3, were slightly lower than either laboratory-held Mytilus or 
those collected from the polluted area at Station 2. However, the overlap in Cd 
values between the three sites renders differences insignificant (Figure 3-4). 

Lead, in those four animals from the polluted area (Station 2) having 
detectable levels, was higher than those levels detected in the laboratory-held 
animals, and the single animal having detectable levels from Station 3. 
However, due to the low number of sampling points, statistical significance 
cannot be established for these data (Figure 3-4). 

Nickel levels in Mytilus from Station 2 are significantly higher than those 
measured in laboratory-held animals. Mytilus from Station 3, while not being 
significantly different from Station 2, or laboratory-held animals, clearly fall in 
a range midway between those polluted and clean areas. A gradient of Ni, with 
highest levels in the polluted area of the Bay, diminishing in the mid-Bay, 
having lowest levels in the lower Bay, is demonstrated by these data. It is of 
interest to note that only 19 of the 41 laboratory-held animals had detectable 
levels of Ni. 


31 



jjg metal/gm tissue dry weight 


100 


Ni 


10.0 


-- U 


.0 


Cu 


I 


T I 


u 


Pb 


I 


L - 
U 


Cd 


Cr 


u 


u 


V 


u 


Zn 


I 


L U S J 


0.1 


L : Laboratory held animals at time = 0 

U : Animals held in unstressed area (Sta. 3, Fig. 3-1 ) 
for 4 week exposure 

S: Animals held in stressed area (Sta. 2, Fig. 3-1) 
for 4 week exposure 


1000 


100 


10.0 


.0 


Note: Bar lines, when present, indicate one standard deviation on either side 
of the mean; otherwise total range is indicated. 


Figure 3-4. Metal levels in Mytilus edulis introduced into 
stressed and unstressed environments, Narragansett Bay, Rl, 

September-October, 1976. 


32 
































Jjg metal/gm tissue dry weight 


100 


Ni 


10.0 


3 1 


1.0 - 


Cu 


3 4 


0 


Pb 


0 


I 


Cd 




-- 




13 4 
0 


Cr 


V 




3 -- 


3 4 


0 


Q; Laboratory held animals at time = 0 

3: Animals held at Sta. 3, Fig. 3-1 for 

3 week exposure 

4: Animals held at Sta. 3, Fig. 3-1 for 

4 week exposure 


1000 


Zn 


0 3 


4 - 100 


10.0 


0.1 


.0 


Note: Bar lines, when present, indicate one standard deviation on either side 
of the mean; otherwise total range is indicated. 


Figure 3-5. Metal levels in Mytilus edulis indicating uptake from 
time=0 to time=4 weeks, Narragansett Bay, Rl, 
September-October 1976. 


33 







































Table 3-1. Metal Levels in Mytilus Edulis 



Cd 

Pb 

Ni 

Cu 

Cr 

V 

Zn 

Lab-held 

2.89 

9.7 

5.7 

8.5 

2.35 

10.7 

170 

41 specimens 

.78 

1.9 

1.6 

2.2 

1.52 

4.3 

52 

(time = 0) 

(40) 

(10) 

(19) 

(41) 

(41) 

(22) 

(41) 

Stressed 

3.04 

12.6 

15 

19 

3.76 

10.5 

199 

10 specimens 

.65 

10.8- 

3.69 

2.8 

.69 

4.7- 

83 



15.5 




11.4 


(time = 4 weeks) 








(10) 

( 4) 

(10) 

(10) 

(10) 

( 5) 

(10) 

Unstressed 

2.42 

7 

9.1 

12 

2.53 

8.4 

149 

10 specimens 

.47 

7 

2.7 

1.4 

.50 

6.4- 

22 







11.5 


(time = 4 weeks) 








(10) 

( D 

(10) 

(10) 

(10) 

( 5) 

(10) 


Metal levels in Mytilus edulis expressed above in ug/g dry weight as: 


Mean 

Standard deviation or range 

(no. of organisms above detection limit) 


Copper levels in Mytilus are significantly higher in that group of animals 
from polluted Station 2 than either Station 3 or laboratory-held animals. 
However, the latter two animal groups have statistically similar levels. Copper 
data show a sharp drop from the polluted area to the levels in both lower Bay 
groups (Figure 3-4). 

Chromium levels in Mytilus from the three stations are not significantly 
different (Figure 3-4). 

Vandium levels were above detection in only about one-half of the 
individuals collected from each station. While the data are too sparse for 

statistical analysis, no difference between the levels at the three stations is 
apparent (Figure 3-4). 

Variability in levels of Zn is so great within each station that meaningful 
comparison between stations is not possible. 


34 








After a three-week period, Mytilus from polluted Station 2 had accumulated 
significantly higher levels of Ni and Cu than had Mytilus from the clean Bay 
stations (Figure 3-5). Similarly, Station 2 values after three weeks were 
significantly higher than values from control animals sacrificed at time zero. 
However, levels remained the same in all animal groups after one additional 
week of exposure. 

DISCUSSION 

Currently, Mytilus edulis is being used as an indigenous biological monitor 
for a variety of materials including petroleum hydrocarbons, chlorinated 
hydrocarbons, and transuranics, in addition to trace metals in coastal waters of 
the United States (5). Similar activity is underway in the United Kingdom and 
Germany as well. Because of its ubiquitous distribution, Mytilus edulis is being 
considered as an international monitoring organism. 

To date, the use of Mytilus edulis appears to be limited to that of an 
indigenous monitor. We are not aware of an approach similar to that reported 
here where Mytilus is used as an introduced biological monitor. 

The results indicate that Mytilus as an introduced species does reflect 
elevated levels of metals previously observed in the polluted section of 
Narragansett Bay, in the sediments and the indigenous mollusc, Mercenaria 
mercenaria. Nickel and copper were concentrated to significantly higher levels 
in Mytilus introduced into the polluted section of the Bay compared to those 
introduced in the clean lower Bay, and those held in our laboratory 
flow-through system. Lead levels were observed to be higher in animals from 
collecting Station 2, than in either collecting Station 3 or the laboratory; 
however, statistical significance was not established due to the small data set. 
Cadmium, chromium, vanadium, and zinc were not reflected at higher levels in 
Mytilus from the polluted area. 

With one exception, that being in the case of Cd, these results reflect those 
reported by Phelps and Myers (5) for Mercenaria collected in the same parts of 
Narragansett Bay. In that study, Mercenaria from the polluted area were shown 
to concentrate to higher levels, but not depurate, Cd, Pb, Ni, and Cu compared 
to lower Bay animals. No differences were observed in levels of V and Zn 
between the two groups of Mercenaria before or after depuration. Thus, 
Mytilus edulis , when used as an introduced monitor, is demonstrated to reflect 
metal levels observed in the major resident or indigenous species in three out of 
the four metals of note. 

Higher metal accumulations were established after three weeks of 
monitoring by Mytilus introduced in the polluted section of the Bay for Ni and 


35 





Cu. Higher levels of Pb appeared after four weeks of exposure. The relatively 
quick response time established in the case of Ni and Cu represents an obvious 
advantage to the use of Mytilus as a biological monitor. For these metals, 
Mytilus quickly reflects a situation which effects the long-lived indigenous 

species, Mercenaria . 

These results encourage further study. Our three specific goals listed in the 
Introduction have been answered in the affirmative: 


1. Introduced Mytilus from polluted areas reflect elevated levels of metals, 
as did sediments and Mercenaria reported in previous work. 

2. Mytilus displays a relatively short response time in accumulating 
elevated metal levels — three weeks in the case of Ni and Cu, and four 
weeks in the case of Pb. 

3 . Mytilus, the introduced biological monitor, took up three of the four 
metals previously demonstrated to have been accumulated and retained 
by the resident species, Mercenaria. 

The results reported here are based on data collected when the annual 
temperature cycle was declining toward winter levels. This fact may account 
for the leveling-off of Ni and Cu observed between weeks three and four at 
Station 2. 

Further studies along the transect in Narragansett Bay, and in other 
comparable areas, including a complete annual temperature cycle, are being 
carried out to supplement the knowledge gained in this study on the use of 
Mytilus edulis as an introduced biological indicator of man’s impact on the 
environment. 

REFERENCES 

1. Phelps, D.K., G. Telek, and R. L. Lapan, Jr. 1975. Assessment of Heavy 
Metal Distribution Within the Food Web in Marine Pollution and Waste 
Disposal. Pearson and Frangiapane, editors. Pergamon Press. 

2. Phelps, D.K. and A. Myers. 1978. Transect Studies in Narragansett Bay, R. I. 
Manuscript. 

3. Farrington, J.W. and J.G. Quinn. 1973. Petroleum Hydrocarbons in 
Narragansett Bay. I. Survey of Hydrocarbons in Sediments and Clams. Est. 
and Coastal Mar. Sci. 1:71:79. 


36 


4. Phelps, D.K. and A. Myers. 1977. Ecological Considerations in Site 
Assessment for Dredging and Spoiling Activities in Ecological Research 
Series, EPA-600/3-77-083, 266-286. 

5. Goldberg, E. 1978. Annual Report, Mussel Watch Program. EPA Contract 
No. R-80421 501. 


37 


TRACE METAL SPECIATION AND TOXICITY 
IN PHYTOPLANKTON CULTURES 


F.M.M. Morel, N.M.L. Morel, D.M. Anderson, 
D.M. McKnight and J.G. Rueter, Jr. 
Division of Water Resources 
and Environmental Engineering 
Civil Engineering Department 
Massachusetts Institute of Technology 
Cambridge, Massachusetts 02139 


ABSTRACT 

The toxicity of trace metals to phytoplankton has been demonstrated to 
depend on metal ion activities. The various chemical processes that control 
metal speciation, and thus activities in aquatic systems, are inorganic 
complexation, chelation, precipitation and adsorption. For example, the 
activity of metals such as mercury, cadmium or lead are controlled in saline 
waters of low organic content by the formation of chloride and bromide 
inorganic complexes. For mercury, this is also the case in typical 
phytoplankton culturing media. Artificial chelating agents permit convenient 
manipulation of metal ion activities in algal toxicity experiments. However, 
kinetic phenomena can result in transient peaks in metal ion activities and lead 
to large overestimations of toxicity. The release of metal complexing agents by 
algae is not expected, in general, to affect markedly the chemistry of metals in 
highly chelated artificial media except in cases of high specific affinity. The 
greatest complication in interpretation of photoplankton toxicity experiments 
arises from the presence of solids in the culture medium. These can precipitate 
during the preparation of the medium, or as a result of the pH increase due to 
photosynthetic carbon uptake. The kinetics of precipitation of these solids, 
their aging and the adsorption of trace metals on their surface, lead to 
variations in metal activities that are difficult to quantify, and do not permit 
proper assessment of the toxic effects. Understanding the global aquatic 
chemistry of trace metals in algal culture media, is a sine qua non prerequisite 
to proper design and interpretation of toxicity experiments. 

INTRODUCTION 

Using copper as the principal example, this study aims at establishing a 
chemical framework for the study of laboratory and natural processes involving 
trace metal toxicity to phytoplankton. If it is a reasonable assumption that the 


38 


mechanisms of metal toxicity are similar in many phyla, this framework should 
be useful for toxicity studies with many aquatic organisms, and can serve as the 
general basis for design and interpretation of such studies. 

Although the importance of both organic complexing agents and trace 
metals in phytoplankton cultures has been recognized for some time, the 
critical role played by the speciation of trace metals in controlling their 
toxicity and availability to algae has just begun to be understood. It has now 
been established that it is the activity of the free ions, rather than the total 
metal concentrations, which determine the toxicity of metals to 
phytoplankton (46, 2). The study of the chemical processes which govern the 
activity of a given trace metal in culture media becomes then a prime area of 
concern to phycologists interested in metal toxicity experiments. An 
enumeration of these processes includes inorganic complexation, chelation, 
precipitation and adsorption. In addition, indirect chemical effects involving 
several interacting chemical species in the medium can influence trace metal 
activities in unobvious ways. In this study, these basic principles of Aquatic 
Chemistry (43) that apply to metals in phytoplankton cultures will be 
discussed systematically. 

There are but a few free metal activities that can be experimentally 
measured in the range and under the conditions of interest. There are also few 
metallic complexes which can be analytically determined in chemical systems 
as complex as culturing media. Henceforth, theoretical equilibrium calculations 
will be used throughout this paper to assess metal speciation and activities. The 
assumption of equilibrium is a reasonable one when the proper precautions are 
taken during medium preparation. Thermodynamic calculations provide, then, 
a convenient means of illustrating the critical chemical principles, even if they 
have inherent uncertainties. Complications introduced in the chemistry of the 
system by kinetic phenomena, or by the influence of the algae, will be 
discussed for each of the examined processes. 

Copper has been the metal of choice in studies of metal toxicity of 
phytoplankton because it has been postulated that cupric ion toxicity might 
play a role in the ecology of phytoplankton in some natural waters (6, 37, 7, 
10). In keeping with this situation, this paper will focus, albeit not exclusively, 
on copper which provides a rather good example for metal speciation and 
toxicity in phytoplankton cultures. 

EQUILIBRIUM SPECIATION OF METALS IN CULTURING 
MEDIA 

Before studying in detail the role of chemical processes in controlling metal 
speciation, it seems useful to examine what metal species are expected to be 
important in typical culturing media. 


39 







In artificial media where the analytical concentrations of the components 
are precisely known, the exact composition of the system, including all soluble 
and insoluble species of the various metals and their activities, can be 
computed if a state of equilibrium or partial equilibrium is established (43). It 
should be underlined that such calculations of chemical speciation depend on 
correct identification of the principal species, and knowledge of the 
corresponding equilibrium constants. The calculations presented here were 
performed with the computer programs, REDEQL (25, 21) and MINEQL(49) 
which contain a list of possible species, and a selection of the necessary 
thermodynamic constants from a variety of sources (36, 38, 33). 

Results of chemical equilibrium computations of three media recipes, the 
freshwater medium WC (11) and the seawater media F/2 (12) and Aquil (26), 
are shown in Table 4-1. Possible adsorption processes are not considered in 
these calculations. Note that some heavy metals (Pb, Cd, Hg, Ni, Co, Cr) which 
are not part of the recipes, have been added in trace amounts (10‘^M) to 
illustrate how they would be speciated if they were present as contaminants in 
the media. Such low metal concentrations affect the rest of the chemical 
systems negligibly. Heavy metal speciation in all media is completely 
dominated by the chelation with ethylenediaminetatraacetate (EDTA) which is 
included in the recipes for the very purpose of chelating metals. An important 
exception is mercury, which, according to the computations, is present entirely 
as chloride complexes in F/2 and Aquil and half as hydroxide species in WC. 
For all metals, the free ion activities are several orders of magnitude smaller 
than their total concentrations. In all media, iron and manganese oxides and 
calcium phosphate (hydroxylapatite) are computed to precipitate at 
equilibrium. Calcium carbonate (calcite) is also shown to be saturated in the 
seawater media. Actual precipitation of these various solids is dependent on 
kinetic processes as will be discussed later. 

The trace metal chemistry of such culturing media can be grossly affected by 
the presence of algal cells due to metal uptake. For example, typical values for 
the uptake of copper by phytoplankton are in the range 10' 1(5 to 10'^ 
moles/cell (44, 35, 16). With the algal densities and the copper concentrations 
commonly used, a sizeable part of the total concentration of copper in the 
medium can thus be taken up by the cells. This underlines the necessity of 
“buffering” metal ion activities in toxicity experiments in order to render these 
activities relatively insensitive to total metal concentrations. The use of various 
chelating agents for this purpose will be discussed later. 

INORGANIC COMPLEXATION 

It has been observed in several instances that the toxicity of metals such as 
lead, cadmium, mercury or silver to a variety of organisms, from bacteria to 


40 


Table 4-1. Equilibrium Trace Metal Speciation in Typical Algal Growth Media 



2: 

m 

I 

o 


CM 


CM 

u- 



CM 


II 

< 

H 

Q 

UJ 

U 



41 















fish, decreases with increasing salinity of the water (45). One possible 
explanation for such a phenomenon is the decrease in metal ion activity 
resulting from the formation of ion pairs with the major anions of seawater. 
Figure 4-1 shows, for example, how the speciation of mercury and the activity 
of the mercuric ion vary in function of salinity in the medium F/2. As salinity 
increases the bromide complexes of mercury replace the EDTA chelate as the 
major species, followed by the chloride complexes as the salinity approaches 
that of seawater. In natural systems, in the absence of strong chelating agents, 
the same phenomenon would extend to other metals such as lead and 
cadmium. Table 4-2 illustrates this point by giving the major species of the 
various metals in Aquil where EDTA has been reduced to 10'^M. Besides the 
chloride complexes, a number of carbonate (Cu, Pb), sulfate (Zn, Mn, Co) and 
hydroxide (Zn, Pb, Co, Cr) complexes become significant. Because the kinetics 
of formation of the various inorganic complexes of metals are typically fast 
(43), equilibrium is a good assumption in this instance, and the thermodynamic 
calculations should give accurate values of metal activities. 

The role of carbonate complexation in decreasing the toxicity of metals in 
unchelated media has been verified for copper on Daphnia magna (3), for 



Note: Top: mercuric complex as a percent of the total mercury (10* 9 M); 

B °^rrvr a tHe activity of the mercur ic ion. All other trace metals remain bound 
to EDTA throughout the salinity range. SW represents seawater, salinity 33ppt. 


42 









Table 4-2. Speciation of Trace Metals in Aquil with 
EDTA Concentration Reduced to 10' 8 M. 



Major Species 

Percent of Total Metal 

Iron 

FE(OH) 3 (S) 

100 % 

Manganese 

MnO^(S) 

100 % 

Copper 

Cu2+ 

1 % 


CuEDTA 

87% 


CuCOo 

12 % 

Cadmium 

Cd2+ 

3% 


CdCI 3 + 

39% 


CdCI 2 

42% 


CdEDTA 

6% 

Zinc 

Nz2+ 

60% 


ZnSO^ 

8% 


ZnCI 

3% 


ZnCI 3 ' 

1 % 


ZnEDTA 

27% 


ZnOH+ 

1 % 

Nickel 

NiEDTA 

99% 

Mercury 

HgCI 4 2- 

87% 


HgCI 3 

13% 

Lead 

PbC0 3 

21 % 


PbCI 3 

50% 


PbCI 2 

5% 


PbCI+ 

3% 


PbEDTA 

20 % 


PbOH+ 

1 % 

Cobalt 

Co2+ 

51% 


C 0 SO 4 

11 % 


CoCI+ 

21 % 


CoEDTA 

15% 


CoOH 

2 % 

Chromium 

CrEDTA 

57% 


Cr(OH) 4 - 

40% 


Cr(OH) 2 + 

3 % 


copper on some fishes (31), and for cadmium on a grass shrimp (45). Although 
there has been no experiment reported to date that provides direct evidence for 
the importance of inorganic complexation in controlling metal toxicity to 
phytoplankton, this result can be inferred from data with these other 
organisms, and from the general demonstration that metal ion activities are the 
important parameters of toxicity to algae. 


43 





Through uptake of carbon dioxide for photosynthesis, algae can modify the 
inorganic species of metals by decreasing the total concentration of carbonate 
in the system and increasing the pH. As pH increases, the hydroxyl ion activity 
increases and so does the importance of metal hydroxide complexes. The e ect 
on the carbonate ion activity and on the metal carbonate complexes is less 
straightforward, and depends on the original pH of the medium. In seawater 
media (pH 8), and in freshwater media around neutral pH, the carbonate 
complexes will increase with C0 2 uptake due to the predominance of the 
resulting pH increase over the total carbonate decrease. Such variations in 
metal chemistry can be alleviated by bubbling air in the cultures, thus insuring 
a steady concentration of carbonate in the medium. 

CHELATION 

The history of the development of artificial culturing media for algae is in 
part that of the replacement ot “growth factors” and soil extracts by 
chelating agents (17). The exact role of these chelating agents in promoting 
algal growth has been a subject of some controversy (6, 14). It is now well 
established that they do control the toxicity of various heavy metals - copper 
in particular (46). Whether they also increase the availability of some metallic 
nutrients — chiefly iron — is yet unproven. Figure 4-2 shows the percentage of 
chelated metal and the metal activities in Aquil (with contaminant metals) as a 
function of the concentration of EDTA, by far the most widely utilized 
chelating agent in algal media. Note that the order in which the metals are 
chelated by EDTA is not simply related to either the metal ion activities or 
their affinities for EDTA (FE>Cr>Cu>Ni>Pb>Zn, Cd, Co). 

Other chelating agents which are commonly used include nitrilotriacetate 
(NTA), citrate and various amino acids. “Tris” (tris(hydroxymethyl)amino 
methane) commonly used as a pH buffer for biological experiments has 
received much use in recent studies of copper toxicity to phytoplankton (44). 
Used in conjunction with EDTA which chelates the other metals at a very low 
concentration, Tris permits a convenient manipulation of the cupric ion 
activity. Figure 4-3 illustrates this point by comparing how the cupric ion 
activity varies with total copper in Aquil (EDTA = 10*^*^M) and in a modified 
Aquil recipe containing Tris (EDTA = lO'^M) and Tris = 10'^M). Around 
[Cu 2 + ] = 10*^M, where many toxicity studies are run, the cupric ion activity 
in the Tris medium is less sensitive to variations in total copper concentration 
than in the EDTA medium. However, with the proper precautions, both media 
yielded the same results in a study of copper toxicity to Gonyaulax tamarensis 
( 2 ). 


44 



B 



Figure 4-2. The effect of EDTA on the speciation of metals 

in Aquil. 


Note: Total metal concentrations are given in Table 4-1, with Pb, Cd, Hg, Ni, 
Co, and Cr added as contaminants (lO'^M). A) The percent of each metal that 
is chelated (MeEDTA) versus total EDTA concentration, (M); B) Metal ion 
activity (M) versus total EDTA concentration (M). 


45 













Figure 4-3. Computed activity of the cupric ion (M) versus 
total copper concentration for two Aquil recipes. 


Note: Chelated with: A) EDTA plus 10' 3 M Tris and B) 10' 5 - 3 EDTA. 

Although the forward kinetic constants of chelate formation are invariably 
very large, resulting in quasi instantaneous kinetics in simple systems, the 
situation can be very different in systems as complex as culturing media. For 
example, when copper was spiked in Aquil cultures of G. tamarensis, a 
dramatic short term toxic response was observed much above that expected for 
the calculated equilibrium activity of the cupric ion (2). This phenomenon 
which was not observed when Tris replaced EDTA as the major copper 
chelating agent, was attributed to the slow kinetics of the metal exchange 
reaction: 


Cu 2+ + CaY ^ CuY + Ca 2+ 

This appears as a reasonable explanation, since the calcium chelate is the major 
form of EDTA in Aquil and the dissociation is slow. No such phenomenon can 
occur with Tris, whose major species in culturing media are the various 
protonated forms of the ligand. This can be checked directly by monitoring the 
cupric ion activity with a mixed sulfide electrode (34, 13) in chemical systems 
similar to the culturing media. Figure 4-4 presents the results of such an 
experiment, and leaves no doubt as to the slow kinetics of copper reaction with 


46 





120 



•f 

c\j 

o 


o» 

o 


10 


Figure 4-4. Effects of dissociation of Ca-EDTA on short term 
cupric ion activity after addition of lO'^M Cu(N 03 ) 2 - 


Note: Background electrolyte 0.5 m KN 03 , pH 8.25 (2 x 10"^M NaHC 03 
bubbled with air), EDTA, temperature 23°C. A Radiometer 

selectrode (F 3000), an Orion d/j reference electrode, and an Orion pH 
electrode were used. In both experiments, the cupric ion activity (M) was 
calculated using the Nernst equation and data from 10'^, 10'^ and 10'^M 
Cu(N 03)2 solutions at pH 4:00 in 10'^N KNO 3 background electrolyte. A) 
Calcium (10‘^M) in equilibrium with EDTA prior to copper addition; B) No 
calcium present. 


47 











EDTA in the presence of an excess of calcium: an initial peak in cupric ion 
activity is measured by the electrode, and it takes about four hours to 
approach the equilibrium value. Such phenomena have to be taken into 
account when studying the toxicity of metals to any aquatic organism, as 
transient effects can lead to large overestimations of toxicity. 

The release of chelating metabolites has been widely assumed as a 
conditioning mechanism for culture media (39). As is the case for natural 
waters, most of the chemically quantitative work on this topic has focused on 
the synthesis and exudation of iron chelating agents, particularly hydroxamates 
(19, 30). What seems often overlooked is that hydroxamic acids do not chelate 
exclusively iron, and that their binding of other metals can result in sizable 
decrease of these metals’ activities (1). 

By direct potentiometric techniques, extracellular metabolites of algae have 
been characterized in terms of copper complexing capacity and affinity (48). 
According to this work, the ligand produced by the algae under the conditions 
of the experiments is characterized by a constant of approximately unity for 
the reaction: 


Cu" + + HY — = H + + CuY 

If one assumes the ligand to be copper specific, the effect of its release in Aquil 
and Aquil with Tris is shown in Figure 4-5. Note that a significant decrease in 
the cupric ion activity does not begin until the total ligand concentration 
reaches 10'^M, an upper limit for the measured ligand releases. Although 
[Cu“ + ] start decreasing at a slightly lower ligand concentration when the 
copper concentration is elevated, the release of such relatively weak 
complexing ligand has little overall effect on the cupric ion activity in a well 
chelated medium. Ligands, with higher affinity for copper, appear to be 
released by some blue green algae (22). 

In principle, phytoplankton could modify the trace metal chemistry of the 
medium by assimilating artificial chelating agents. However, this potential 
problem is avoided by using EDTA or NTA which have been shown not to be 
assimilated by algae (23). Although photodegradation of EDTA and NTA has 
been reported (40), the light intensities normally used for culturing 
phytoplankton are insufficient to promote it in the laboratory. 

PRECIPITATION 

According to the computations of Table 4-1, the precipitation of several 
solids is calculated to be thermodynamically favorable in typical culturing 
media. Visible precipitates are indeed a common observation of users of algal 


48 



Figure 4-5. The effect of different molar concentrations of 
copper specific metabolite (Y) on the activity of the cupric ion 
for three variations of Aquil medium. 

Note: A) 10‘ 5 - 3 M EDTA plug 10' 6 M Cu; B) 10' 6 3 M EDTA plus 10' 3 M Tris; 
C) 10' 5 3 M EDTA. 

media. This is especially true following autoclaving, which brings about a large 
pH increase by eliminating carbon dioxide from the system. This problem has 
been studied by researchers involved in the design of culturing media (32, 8, 
12, 18, 26). The increase in temperature and pH during autoclaving decreases 
the solubility of calcium carbonate, and results in the precipitation of a 
magnesium rich solid (this suggests the solid to be magnesium calcite, although 
aragonite has been identified in such precipitates). Hydrous oxides of iron and 
manganese can also precipitate under such conditions, depending on the 
chelating agent concentration and the pH reached during autoclaving. When 
such precipitates occur, phosphate becomes largely associated with the solid 
phase, presumably in some calcium precipitates (apatite or CaHPC^), or as an 
adsorbate on the various solids. Depending on the initial concentration of 
silicic acid and on the nature of the container, which can increase the silicate 
concentration of the solution by dissolution, some amorphous or crystalline 
form of Si 02 can form in the medium. 


49 






LEAD OXIDE 
COBALT HYDROXIDE 


MAGNESIUM HYDROXIDE 
STRONTIUM SULFATE 


IRON HYDROXIDE 
CALCIUM CARBONATE 


MANGANESE OXIDE 


CALCIUM PHOSPHATE 

i ■ 1 ■ 1 J-J-- » 

6 7 8 9 10 pH 


Figure 4.6. Saturation pH for various metal solids in Aquil. 


These various solid formation processes are dependent on kinetic factors 
which are controlled by the particular temperature and pH regime of the 
medium. These, in turn, depend on the conditions and duration of autoclaving, 
as well as on the size of the containers and the mixing conditions. Precipitates 
are rarely seen with filter sterilization. Figure 4-6 shows the onset of saturation 
for various solids as pH is increased in the medium Aquil, normally designed to 
avoid precipitates. None of the four solids that are saturated at pH = 8 in Aquil 
are actually seen to precipitate, even after autoclaving if the volumes are kept 
smaller than 100 ml. If larger volumes are autoclaved, immediate bubbling with 
carbon dioxide prevents precipitation. Avoidance of calcium carbonate 
precipitate is very important for maintaining iron and manganese in solution, as 
the presence of CaCO^(s) will catalyze the formation of hydrous oxides of 
manganese and iron (43). For other trace metals the formation of these 
precipitates creates difficulties mostly through adsorption processes (see next 
section). 

For toxicity studies, trace metals are sometimes introduced in algal cultures 
in excess of the chelating agent concentration. Precipitates are then often 
expected to lorm mostly oxides, hydroxides and carbonates, depending on the 
metal. For example, a hydroxide (Cu(OH) 2 ), an oxide (CuO, tenorite) when a 
carbonate (Cu 2 C 03 ( 0 H) 2 , malachite) become quickly saturated in Aquil when 
copper exceeds the EDTA concentration. Although the hydroxide is not the 


50 








thermodynamically stable form, it probably is the one which forms initially in 
the medium for kinetic reasons. Regardless of the precise nature of the solid, 
good agreement has been obtained between calculated and measured copper 
concentration in the solid phase in Aquil medium with a high EDTA 
concentration, 2 hours after addition of excess copper (29). It is worth noting 
that the precipitate was very finely dispersed, and that centrifugation was 
necessary to separate it from the aqueous phase. Ignorance of the formation of 
a precipitate can obscure completely the meaning of otherwise well controlled 
experiments. In terms of metal ion activity, the situation is complicated by the 
change in the nature of the precipitate which might evolve from an active form 
to a more stable one. In copper saturated media, the cupric ion activity has 
been measured potentiometrically to decrease markedly over 24 hours, the rate 
of decrease becoming very small thereafter (22). Such conditions can create 
large uncertainties in toxicity experiments. 

ADSORPTION 

The common notion that chelating agents make iron available to algae, 
seems to be supported by experiments where addition of iron or EDTA salts 
provide similar growth and carbon uptake enhancement in a variety of algal 
cultures (3). However, aluminium salts have also been observed to enhance 
carbon uptake (24). Following Stumm and Barber (41), it is now a prevalent 
interpretation of such experiments to attribute part, or all of the beneficial 
effect of the metal additions to a scavenging of other toxic metals by 
adsorption on precipitating iron or aluminium hydrous oxides. Figure 4-7 
illustrates the beneficial effect of iron additions to a Pyramimonas culture, and 
demonstrates how iron and copper behave antagonistically under controlled 
conditions (28). The growth rate of Pyramimonas is reduced at a total copper 
concentration of 1.2 10 M, and completely stopped at 4.4 10 M when the 
iron concentration is low (1.2 10'^M). Increasing the iron concentration by a 
factor of 10 completely blocks the toxic effect of the same copper 
concentrations. The question to be resolved is how much of this 
“detoxification” of copper by iron is due to adsorption processes, effectively 
removing the copper from solution and decreasing the cupric ion activity, and 
how much is due to a genuine physiological antagonistic effect at the cellular 
level. In a recent study of the adsorption of copper on hydrous iron oxide in 
seawater (48), it has been observed that under conditions similar to the 
experiments of Figure 4-7, iron adsorbs copper up to a Fe/Cu molar ratio of 
1/3. Adsorption can then certainly account for all of the antagonistic effects in 
the Pyramimonas experiment. What becomes more difficult to explain is the 
lack of antagonistic effect at the low iron concentration (1.2 xlO’^M) since 
even then the highest copper concentration (4.4 x lO'^M) should be entirely 
adsorbed. Note, however, that this is a domain of concentrations where copper 
starts saturating the colloidal iron surface, and there must be a titration effect 


51 


# Cells /mb 



Days 


r e 

C u 

1 .2x10 

' 5 tyl 

1 .2x10 6 M 

Contro 1 

□ 

o 


4 x IO~ 8 M 

A 

1.2 x IO' 7 M 


• 

▲ 

4.4 x ICT 7 M 

■ 


▼ 


Expt. #1 Expt.#2 Expt.#3 

-Experiments #1,2,3 done with inocula from 
different cul tures 
-No EDTA 


Figure 4-7. Antagonistic effects of iron and copper on 
the growth rate of Pyramimonas 1 in artificial seawater medium 
with the usual supplements of F/2 medium except for EDTA 

which is not added. 


Note: Experiments 1, 2, and 3 represent inocula from different cultures. 
"Fresh" iron is FeCl 3 solution prepared the day of the experiment and 
sterilized by filtration. 


52 






















(like that of copper on EDTA in Figure 4-3) where the cupric ion activity 
increases rapidly with increasing copper concentration. Exact quantitification 
of this phenomenon awaits a better mathematical description of adsorption 
processes on hydrous iron oxides in seawater. Despite great recent advances in 
the modeling of adsorption in aqueous systems (50, 42, 15), it is still the least 
quantifiable chemical process in thermodynamic calculations. The presence of 
precipitates in a culture medium modifies its global trace metal chemistry to an 
unpredictable degree. This creates the most common difficulty in interpreting 
experiments on toxicity of metals to a variety of organisms. Note that 
adsorption on the walls of a glass culture vessel is equally hard to predict. 
Choice of container material which minimizes adsorption of solutes is critical 
to the design of trace metal toxicity experiments. 

Adsorption on the surface of algal cells can also be important for the trace 
metal chemistry of the medium in dense cultures. There is, however, no 
practical way to distinguish it from intracellular uptake. The effects of cellular 
uptake processes including adsorption on the cell surface, have been discussed 
earlier. 

INDIRECT CHEMICAL EFFECTS 

The general principles of coordination, precipitation, and adsorption which 
have been discussed heretofore, are readily understood and their importance in 
toxicity studies is usually recognized. What is less often perceived is the global 
interdependency of the chemistry of culture media, the indirect interactions 
(43, 27). For example, upon variations in the total copper concentrations, it is 
natural to relate the observed effects to changes in the cupric ion activity. 
However, as illustrated in Figure 4-8, activities of the zinc and ferric ions are 
also increased when the total copper is augmented in Aquil. Conceivably, any 
or all of these increased activities could be responsible for the observed effects. 
It is then a difficult choice to either maintain all metal activities constant by 
adhoc modification of all analytical concentrations — a method which 
multiplies the work for medium preparation and can create other interpretative 
ambiguities — or to perform the multitude of necesary controls on an already 
arduous series of experiments. Table 4-3 shows how the total metal 
concentrations have to be varied concomitantly with that of copper, to vary 
exclusively the cupric ion activity in Aquil with two EDTA concentrations 
(35). 

The indirect interactions illustrated in Figure 4-8 are almost exclusively 
mediated by EDTA, which chelates all the interdependent metals. In principle, 
a convenient way to avoid the complications created by these interactions is to 
reduce them to a minimum. This can be achieved by uncoupling the system 
using more specific complexing agents. Figure 4-8 shows how the metal 


53 


log (Mn 2 *\ Zn 2 




Figure 4-8. Variations in metal activities (M) of manganese, 
zinc and iron with total copper concentration. 


Note: A) Aquil medium, 10' 5 - 3 M EDTA; B) Aquil with 10' 6 - 3 M EDTA 
10* 3 M Tris. 


54 


-log 

















Table 4-3. Calculation of Total Metal Concentrations Needed 
to Change the Cupric Ion Activity in Aquil While Maintaining 
the Other Metal Activities Constant 
(-log (concentration) or [activity], M) 


(EDTA) t 

[CU 2+ ] 

(Copper)-p 

(Iron)-p 

(Mang)-j- 

(Zinc)y 

(Cobalt)-j- 


8.5 

3.30 

7.00 

7.63 

8.7 

8.6 

3.3 

10.9 

3.70 

4.72 

6.40 

6.49 

6.7 


11.3 

4.0 

4.6 

6.30 

6.40 

6.6 


9.8 

4.35 

6.45 

7.20 

8.30 

8.49 

4.3 

10.9 

4.70 

5.72 

7.20 

7.49 

7.7 


11.3 

5.00 

5.58 

7.15 

7.4 

7.6 


activities vary with total copper in modified Aquil medium containing “Tris”, a 
ligand known to chelate mostly copper (44). Upon variations in copper 
concentration, the other metals are seen to have a much more constant activity 
in Aquil with Tris than in Aquil with only EDTA. 

One of the principal ways by which indirect chemical effects can be initiated 
is through pH variations. For example, pH has an indirect effect on metal 
complexation due to the acid-base properties of the coordinating ligannds. 
Figure 4-9 illustrates this effect for Mn, Cu and Zn in Aquil, with EDTA and 
Aquil with Tris. In this case, Tris mediates a much greater indirect effect than 
EDTA: Zinc and especially cupric ion activities are markedly depressed by 
increasing pH in the Tris medium, while the activities of all three ions remain 
essentially constant in the EDTA medium. Increases in pH, which can be 
brought about by photosynthetic carbon uptake if the aeration of the culture 
is insufficient, can also result in precipitation as illustrated in Figure 4-6. 
Adsorption on the fresh precipitate will follow, resulting in an unquantified 
decrease in the soluble concentration of trace metals. It is apparent that pH is a 
major factor in determining directly and indirectly the activity and toxicity of 
trace metals, and should be monitored regularly in metal toxicity experiments. 

CONCLUSION 

The chemistry of metals in the external milieu of algal cells is only one of 
the determinants of their toxicity. The literature on bacteria and higher cells 
abounds with examples of how the sensitivity of a particular strain or clone to 
a particular toxicant, depends markedly on the physiological status of the cells 
(9, 4). Although it is often recognized that the same situation should apply to 
phytoplankton, this concept has received scant attention in recent algal 
literature. It stands to reason that the previous history of an algal cell, its 


55 






Figure 4-9. Variations in metal activities with pH. 

Note: A) Aquil medium, 10' 53 M EDTA; B) Aquil with EDTA 

10' 3 M Tris. 



56 













nutritional status, and the particular phase of the cell cycle during which the 
experiment is conducted — to name but a few obvious determinants of 
physiological status — must affect its sensitivity to trace metals. Batch culture 
experiments which, so far, have been used principally for metal toxicity 
studies, have inherent restrictions to resolve the importance of these 
physiological factors. Toxicity studies in continuous phytoplankton cultures 
promise to be enlightening in this respect; they also promise to accentuate the 
difficulties in controlling precisely the chemistry of the system. 

It is hoped that the conceptual framework presented here will help in 
designing and interpreting experiments where physiological responses to trace 
metal toxicity are clearly assessed, distinctly from purely chemical effects in 
the growth medium. It is also hoped that this study will help to increase 
phytoplankton physiologists’ awareness of the important chemical processes 
which can affect their studies. It is, for example, surprising that so little 
attention has been paid to the possible importance of phosphate speciation in 
nutrient uptake experiments. Understanding the ecology of phytoplankton 
requires detailed resolution of the cells’ physiological responses to the total 
aquatic chemistry of their environment. 

ACKNOWLEDGMENTS 

We thank S.W. Chisholm for her critical review of the manuscript and R.C. 
Selman for her excellent job in typing the manuscript. This work was funded 
by National Science Foundation grant no. DES75-15023, Environmental 
Protection Agency grant no. R-803738 and the office of Sea Grant in the 
National Oceanic and Atmospheric Administration grant no. 04-6-158-4407. 

REFERENCES 

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Hydroxamatkomplexe (III 1 ). Eisen (III)-Austausch zwischen Sideraminen 
und Komplexonen, Diskussion der Bildungskonstanten der 
Hydroxamatkomplexe. Helv. Chini. Acta. 46:1409-1422. 

2. Anderson, D.M. and F.M.M. Morel 1978. Copper Sensitivity of Gonyaulax 
tamarensis , Lirr.nol. Oceanogr. (in press). 

3. Andrew, R. W., K.E. Biesinger, and G.E. Glass. 1976. Effects of Inorganic 
Complexing on the Toxicity of Copper to Daphnia Magna , Water Res. 
11:309-315. 

4. Baird-Parker, A.C., and R. Holbrook 1971. In: Inhibition and Destruction 
of the Microbial Cell (ed.), W.B. Hugo, Acad. Press. 


57 



5. Barber, R.T., R.C. Dugdale, J.J. Maclsaac, and R.L. Smith. 1971. 
Variations in Phytoplankton Growth Associated with the Source and 
Conditioning of Upwelling Water, Inv. Peso. 35(1): 171. 

6. Barber, R.T. 1973. In: Trace Metals and Metal-Organic Interactions in 
Natural Waters, P. C. Singer (ed.), Ann Arbor Science, Mich. 

7. Davey, E.W., M.J. Morgan, and S.J. Erickson. 1973. A Biological 
Measurement of Copper Complexation Capacity of Seawater, Limnol. 
Oceanogr. 18:993-997. 

8. Droop, M.R. 1961. Some Chemical Considerations in the Design of 
Synthetic Culture Media for Marine Algae. Botanica Marina. 2:231. 

9. Farwell, J.A., and M.R. W. Brown 1971. In: Inhibition and Destruction of 
the Microbial Cell (ed.), W.B. Hugo, Acad. Press. 

10. Gachter, R., K. Lum-Shue-Chan, and Y.K. Chau. 1973. Complexing 
Capacity of the Nutrient Medium and its Relation to Inhibition of Algal 
Photosynthesis by Copper, Schweizerische Zeitschrift fur Hydrologie. 
35:252-260. 

11. Guillard, R.R.L., and J. Lorenzen. 1972. Yellow-Green Algae with 
Chlorophyllide-C, J. Phycol. 8:10-14. 

12. Guillard, R.R.L., and J.H. Ryther. 1962. Studies on Marine Planktonic 
Diatoms I. Cyclotella nana Hustedt and Detonula Confervacea (Cleve) 
Gran, Can. J. Microbiol. 8:229-239. 

13. Hansen, E.H., C.G. Lamn, and J. Rizicka 1972. Anal. Chim. Acta. 59:403. 

14. Jackson, G.A., and J.J. Morgan 1977. Trace Metal-Chelator Interactions 
and Phytoplankton Growth in Seawater Media, Limnol. Oceanogr. (in 
press). 


15. James, R.O., and T.W. Healey. 1972. Adsorption of Hydrolyzable Metal at 
the Oxide-Water Interface, J. Colloid and Interface Sci. 40:42. 

16. Jensen, A., B. Rystad, and S. Melsom. 1976. Heavymetal Tolerance of 
Marine Phytoplankton II. Copper Tolerance of Three Species in Dialysis 
and Batch Cultures, J. Exp. Mar. Biol. Ecol. 22:249-256. 


58 


17. Johnston, R. 1964. Seawater, the Natural Medium of Phytoplankton. II. 
Trace Metals and Chelation, and General Discussion, J. Mar. Biol. Assoc. 
U.K. 44:87-109. 


18. Jones, G.E. 1967. Precipitate from Autoclaved Seawater, Limnol. 
Oceanogr. 12:165-167. 

19. Lange, Willy. 1974. Chelating Agents and Blue-Green Algae, Can.J. 
Microbiol. 20:1311-1321. 

20. Lockhart, H.B., Jr., and R.V. Blakeley. 1975. Aerobic Photodegradation of 
Fe(III)-(Ethylenedinitrilo) tetraacetate (Ferric EDTA), Environ. Sci. and 
Technol. 9:1035-1038. 


21. McDuff, R.E., and R.M. Morel. 1972. T.R. #EA-73-02, Keck Laboratories, 
California Institute of Technology, Pasadena, Calif. 

22. McKnight, D.M. Unpublished results. 

23. McNeel, T. Unpublished results. 

24. Menzel, D.W., E.M. Hulbert, and J.H. Ryther. 1963. Effects of Enriching 
Sargasso Sea Water on the Production and Species Composition of the 
Phytoplankton, Deep-Sea Res. 10:209-219. 

25. Morel, F.M., and Morgan, J.J. 1972. A Numerical Method for Coupling 
Equilibrium in Aqueous Chemical Systems, Envir. Sci. and Technol. 6:58. 

26. Morel, F.M., J.C. Westall, J.G. Rueter, and J.P. Chaplick. 1975. Description 
of the Algal Growth Media “Aquil” and “Fraquil”, Technical Note No. 16, 
Water Quality Lab., Ralph M. Parsons Laboratory for Water Resources and 
Hydrodynamics, Dept, of Civil Engineering, Mass. Instit. of Technol., 
Cambridge, Mass. 

27. Morel, F.M., R.E. McDuff, and J.J. Morgan. 1973. In: Trace Metals and 
Metal-Organic Interactions in Natural Waters, Interactions and Chemostatis 
in Aquatic Chemical Systems, P.C. Singer (ed.), Ann Arbor Sci. 

28. Morel, N.M.L., and F.M. Morel. 1976. Lag Phase Promotion in the Growth 
of Pyramimonas 1 by Manipulation of the Trace Metal Chemistry of the 
Medium, Technical Note No. 17, Water Quality Lab., Ralph M. Parsons 
Laboratory for Water Resources and Hydrodynamics, Dept, of Civil 
Engineering, Mass. Instit. of Technol., Cambridge, Mass. 


59 


29. Morel, N.M.L., J.G. Rueter, and F.M. Morel. 1977. Copper Toxicity to 
Skeletonema Costatum, J. Phycol. (in press). 

30. Murphy, T.P., D.R.S. Lean, and C. Nalewajko. 1976. Blue-Green Algae: 
Their Excretion of Iron Selective Chelators Enables Them to Dominate 
Other Algae, Science 192:900-902. 

31. Pagenkopf, G.K., R.C. Russo, and R.V. Thurston. 1974. Effect of 
Complexation on Toxicity of Copper to Fishes, J. Fish. Res. Bd. Can. 
31:462-465. 

32. Provasoli, L., J.J.A. McLaughlin, and M.R. Droop. 1957. The Development 
of Artificial Media for Marine Algae, Arch. Mikrobiol. 25:392-428. 

33. Ringbom, A. 1963. Complexation in Analytical Chemistry, Interscience. 

34. Ross, J.W. 1969. In: Ion Selective Electrodes, R. Durst (ed.), Nat. Bur. 
Standards (USA) Spec. Pub. 314. 

35. Rueter, J.G. 1977. The Response of Skeletonema costatum to Copper 
Relating to the Question of Medium Conditioning, Master’s Thesis, Mass. 
Instit. of Technol., Cambridge, Mass. 

36. Sillen, L.G., and A.E. Martell. 1964, 1971. Stability Constants, Special 
Publication, No. 17 and No. 25, The Chem. Soc. of London. 

37. Smayda, T.J. 1974. Bioassay of the Growth Potential of the Surface Water 
of Lower Narragansett Bay over an Annual Cycle Using the Diatom 
Thalassiosira pseudonana (Oceanic Clone, 13-1), Limnol. Oceanogr. 
19:889-901. 

38. Smith, R.M., and A.E. Martell. 1976. Critical Stability Constants, Vol. 4, 
Inorganic Complexes, Plenum, N.Y. 

39. Steemann Nielsen, E., And S. Wium-Anderson 1971. The Influence of Cu 
on Photosynthesis and Growth in Diatoms, Physiol. Plant. 24:480-484. 

40. Stolzberg, R.J., and D.N. Hume 1975. Rapid Formation of Iminodiacetate 
from Photochemical Degradation of Fe(III) Nitrilotriacetate Solutions, 
Environ. Sri. and Technol. 9:654. 


41. Stumm, W. 1969. Personal Communication to Barber, Referenced in 
(1973) Barber, Trace Metals and Metal-Organic Interactions in Natural 
Waters, P.C. Singer (ed.), Ann Arbor Science, Ann Arbor, Mich. 


60 


42. Stumm, W., Herbert Hohl, and Felix Dalang 1976. Interaction of Metal 
Ions with Hydrous Oxide Surfaces, Croatica Chemica Acta. 48:491*501. 

43. Stumm, W., and J.J. Morgan 1970. Aquatic Chemistry, Wiley-Interscience, 
New York. 

44. Sunda, W.G. 1975. The Relationship Between Cupric Ion Activity and the 
Toxicity of Copper to Phytoplankton, Doctoral Thesis, Woods Hole 
Oceanographic Instit., Woods Hole, Mass. 

45. Sunda, W.G., D.W. Engel, and R.M. Thuotte 1978. Cadmium Toxicity to 
the Grass Shrimp, Palaemonetes pugio, as a Function of Free Cadmium Ion 
Concentration (to be published in Envir. Sci. and Technol.). 

46. Sunda, W.G., and R.R.L. Guillard 1976. Relationship Between Cupric Ion 
Activity and the Toxicity of Copper to Phytoplankton, J. Mar. Res. 
34:511-529. 

47. Swallow, K.C. 1977. Adsorption of Trace Metals by Hydrous Ferric Oxide 
in Seawater, Ph.D. Thesis, Dept, of Chemistry, Mass. Instit. of Technol., 
Cambridge, Mass. 

48. Swallow, K.C., J.C. Westall, D.M. McKnight, N.M.L. Morel, and F.M. Morel 
1977. Potentiometric Determination of Copper Complexation by 
Phytoplankton Exudates, Limnol. Oceanogr. (in press). 

49. Westall, J.C., J.L. Zachary, and F.M. Morel 1976. MINEQL, a Computer 
Program for the Calculation of Chemical Equilibrium Composition of 
Aqueous Systems, Technical Note No. 18, Water Quality Lab., Ralph M. 
Parsons Laboratory for Water Resources and Environmental Engineering, 
Dept, of Civil Engineering, Mass. Instit. of Technol., Cambridge, Mass. 

50. Yates, D.E., S. Levine, and T.W. Healey 1974. Site Binding Model of the 
Electrical Double Layer at the Oxide/Water Interface, Far Soc. I., J. Chem. 
Soc. 70:1802. 


61 


A SIMPLE ELUTION TECHNIQUE FOR 
THE ANALYSIS OF COPPER IN 
NEANTHES ARENACEODENTATA 


Gerald L. Hoffman and Raymond M. Zanni 
U.S. Environmental Protection Agency 
Environmental Research Laboratory 
South Ferry Road 
Narragansett, R.l. 02882 


ABSTRACT 

It is common practice to dissolve the tissue of marine organisms completely 
with acid prior to metal analysis with atomic absorption. However, it may not 
be necessary to completely destroy the organic matrix with acids prior to metal 
analysis. It has been determined that a simple 5 percent HNO 3 elution of a 
freeze-dried Neanthes arenaceodentata is sufficient to extract Cu quantitatively 
from this marine polychaete. This type of elution, rather than complete 
dissolution, has several advantages when analyzing small (1 mg to 10 mg) 
organisms. The two major advantages are (1) blank values are lower, and 
( 2 ) the technique is less tedious and time consuming. 

INTRODUCTION 

High temperature ashing and/or various acids (HNO 3 , H ^ S 0 4 > HC10 4 ,etc.) 
are generally used to break down, oxidize, and solubilize marine organisms 
prior to metal analysis by atomic absorption. If metal levels are high, and the 
organisms weigh several grams, the techniques of dry ashing and wet ashing are 
usually successful. However, solubilizing individual organisms that weigh 1 mg 
to 10 mg with standard techniques without contaminating the final solutions 
for the element of interest is difficult. 

Matsunaga ( 1 ) has shown that it was possible to extract Hg completely from 
various types of fish muscle with 1 N HC1 containing cupric chloride. In his 
study, no attempt was made to solubilize the fish muscle tissue. Therefore, we 
reasoned that a simple elution with 5 percent HNO 3 might be sufficient to 
extract metals from small marine organisms. The feasibility of extracting Cu by 
this elution technique was tested on the polychaete, Neanthes arenaceodentata. 


62 


EXPERIMENTAL METHODS 
Apparatus 


All atomic absorption analyses were made using a Perkin-Elmer atomic 
absorption unit (Model 360) coupled to a Perkin-Elmer heated graphite 
atomizer (Model No. HGA-2100). The weight measurements of the worms 
were made with a Perkin-Elmer microbalance (Auto balance Model No. 
AD-2Z). Freeze-drying of the worms was accomplished using a Virtis (Model 
No. 10-145MR-BA) lyophilizer. Low temperature ashing of the samples was 
done with a L.F.E. (Model LTA-505) low temperature asher. 

Reagents and Materials 

Ultrex HNO^ was used throughout the analytical elution and dissolution 
procedures. Copper standards were made up from a stock solution of ALPHA 
atomic absorption standard copper. All 2/5 dram polyethylene snap-cap vials 
were acid washed in concentrated HNO^ for two days, soaked in demineralized 
water for two days, and finally rinsed five times with copious quantities of 
demineralized water. The vials were allowed to air dry in a class-100 clean 
bench. 

Procedure 

The 61 polychaete specimens used in this study were raised in the 
laboratory. Complete details of the methods to raise the worms are given by 
Pesch and Morgan (2). Live polychaete samples were removed from the 
seawater tanks with the aid of a nylon brush and rinsed in control seawater for 
approximately one minute, and then placed in precleaned polyethylene vials 
(1.2 ml capacity) fitted with snap-caps. The samples were frozen and then 
freeze-dried for 24 hours. The freeze-dried worms were then weighed. The 
average weight was 8.7 ± 4.7 mg. One ml of 5 percent HNO^ was added to the 
worms in their respective vials. The sample vials were capped and allowed to 
stand at room temperature for two days. The worms were then transferred 
from the first extraction vial (A) to precleaned tared vials (B) with the aid of a 
teflon fiber. The tared vials (B) containing the wet acid leached worms were 
again weighed. One ml of 5 percent HNO^ was added to the B vials. The (B) 
vials were capped and allowed to stand at room temperature for two days. The 
worms were again transferred to pretared vials (C) and weighed wet. The 
worms were then freeze-dried and weighed again. During these transfer steps 
care was taken so that the worms did not disintegrate. The insoluble worm 
carcasses were then destroyed by low temperature ashing. The freeze-dried 
worms were inserted into teflon beakers (10 ml capacity) and ashed for 24 
hours at the following conditions: 0 o flow 50 cc/min; and RF power of 50 


63 


watts. After ashing, the inorganic residue was transferred back into the (C) 
vials. The transfer was facilitated by adding 0.1 ml of ultra-pure concentrated 
HNO 3 to the teflon beaker and slowly picking up the inorganic residue into the 
drop of HNO. as it was rolled around the inside of the beaker. The HNO 3 does 
not wet the inside of the beaker and can be quantitatively transferred into the 
polyvial. The (C) vials were capped and allowed to stand at room temperature 
for several days to insure dissolution of the particulate residue. One ml of 
demineralized water was added to the (C) vials after the dissolution period. The 
final acid concentration in the (C) vials was approximately 1.6 N in HNO 3 . All 
three vial solutions (A, B and C) were then analyzed for their Cu content. 

RESULTS AND DISCUSSION 

Three of the (A) vial solutions were monitored for increases in Cu content 
during the first 15 hours of the extraction process. This data is plotted in 
Figure 5-1. It can be seen from Figure 5-1 that the extraction appears to be 
fairly rapid, and approaches a constant value at 15 hours. This particular data 
was the major reason for selecting a two-day elution time for the rest of the 
worms processed. 



minutes hours 


TIME 

Figure 5-1. Extraction of Cu from Neanthes arenace odentata 
with 5 percent HNO 3 function of time. 

64 







The weight measurements made during the various processing steps allowed 
the calculation of the amount of 5 percent HNO^ transferred with each worm 
from vial (A) to vial (B), and from vial (B) to vial (C). Therefore, the amount 
of solubilized Cu transferred during the transfer steps could be calculated. The 
amount of 5 percent HNO^ transferred from the (A) vial to the (B) vials ranged 
between 7 and 20 percent of the 5 percent HNO^ present in the (A) vials. 
Figure 5-2 shows the calculated mass of Cu that should be present in the (B) 
vials versus the measured mass of Cu present in the (B) vials. The solid line 
represents a perfect correlation, and is not the calculated regression line. This 
plot shows that there is very little copper that cannot be accounted for in the 
second elution that has not been transferred from the first solution. 

Figure 5-3 shows a plot of the original freeze-dried weights of the worms, 
versus the freeze-dried weights of the worms after two elutions in 5 percent 
HNO 3 . It is interesting to note that the worms lost 50.5 ± 7.2 percent of their 
weight with the two elutions. The Na concentrations were measured in all 
samples, and indicated that only about half of the freeze-dried weight loss 
could be attributed to the loss of solubilized NaCl. Most of the unexplained 
weight loss probably comes from solubilized organic matter. On a qualitative 
basis, this was confirmed by the color of the (A) and (B) solutions which were 
pale yellow in color, and contained very surface active compounds. Even 
though the worms may have been slowly dissolving in the 5 percent HNO^, 
•only a few of the worms had broken down into two or more pieces, and 
generally from a physical appearance looked unchanged. 



Figure 5-2. Calculated mass of Cu in the (B) vials versus the 
measured mass of Cu in the (B) vials. 

65 



14 


o» 

E 


10 


C D 
IxJ 
£ 

Q 

LU 

cr 

a 

Ixl 

N 

Ixl 

Ixl 

o: 

Lx 


o 

o 

Ixl 

CO 


2nd weight 
— I st weight 


2 - 


X 100 = 50.5 t 7.5% • 






•y. 


A* • • 


• • 


/ • 
•f • 


8 


12 


16 20 


24 


28 


FIRST FREEZE DRIED WEIGHT (mg) 

Figure 5-3. First freeze-dried weight versus the second freeze- 
dried weight of Neanthes arenaceodentata samples. 


The elution efficiency (EE) of the First extraction, versus the total Cu 
present in the worms, was calculated by the following equations: 

r 

EE%= -IMxlOO 


where Cj = 

C 1M + ( C 2M ' C 2C) + ^ C 3M ' C 3C) 

an d Cy 

= Total mass of Cu 

C 1M 

= Measured Cu mass in the (A) vial 

C 2M 

= Measured Cu mass in the (B) vial 

C 3M 

= Measured Cu mass in the (C) vial 

C 2C 

= Calculated Cu mass in the (B) vial 

C 3C 

= Calculated Cu mass in the (C) vial 


The calculated EE% of Cu for this set of samples was 97.8 ± 1.8 percent. 
Therefore, lor all practical purposes of analysis for environmental samples, the 
first extraction of the worms with 5 percent HN0 3 is essentially complete for 


66 






Using this elution technique it is possible to extract and analyze large 
numbers of worms for copper very simply, since only the first extract need be 
analyzed. The methods and conditions used in this study should not be 
considered to be the ultimate in elution of metals from marine organisms. It 
may be possible to use other dilute acids (i.e. HF, HC1, H^SO^, etc.) that may 
be more effective for the elution of other metals from different species. If 
elution rather than total destruction of the animal matrix is desirable, then a 
thorough study should be made of the effectiveness of the procedure chosen. 
Dilute acid elution has several advantages over complete destruction of the 
sample matrix. The first and most important is the potential for providing 
lower blanks. The second is the simplicity involved, which allows processing 
100 small organisms in approximately 8 contact hours. The worms need not be 
removed from their respective extraction vials prior to analysis, since they sink 
to the bottom of the vial and do not interfere with HGA atomic absorption 
analysis. The samples do not need to be analyzed within any constrained time 
frame. Some samples have been analyzed repeatedly over a period of several 
months, and have shown no tendency for a concentration change with respect 
to Cu. The worms also do not decompose over this period of time, as they 
appear to be permanently preserved. At this time, we have used this elution 
technique to analyze over 1000 small marine organisms for Cu. 

ACKNOWLEDGMENTS 

We thank Ms. Carol E. Pesch and Mr. Douglas Morgan for providing the test 
animals for this study, and Dr. P. Rogerson for consultation during the course 
of this work. 

REFERENCES 

1. Matsunaga, K., T. Ishida, and T. Oda 1976. Extraction of Mercury from Fish 
for Atomic Absorption Spectrometric Determination. Anal. Chem. 48:1421. 

2. Pesch, C.E., and D. Morgan. 1977. Influence of Sediment in Copper Toxicity 
Tests with the Polychaete Neanthes arenaceodentata. Submitted for 
publication to Water Research. 


67 


GEOCHEMISTRY OF FOSSIL FUEL 
HYDROCARBONS IN MARINE SEDIMENTS: 

SELECTED ASPECTS 


John W. Farrington 
Associate Scientist 
Department of Chemistry 
Woods Hole Oceanographic Institution 
Woods Hole, Massachusetts 02543 


ABSTRACT 

Three investigations are described which illustrate recent advances in 
analytical chemical and geochemical research on fossil fuel hydrocarbons in the 
marine environment. 

First: The application of quantitative gas chromatography-mass 

fragmentography to measure selected aromatic hydrocarbons in marsh and 
coastal sediments. Instrument precisions of 2 to 3% for 50 x 10’^ g of 
naphthalene and 1 -methylnaphthalene are achieved. The detection limit for 
naphthalene (signal/noise ratio of 2:1) is estimated to be 5 x 10’^ g/g dry 
weight of sediment with 25-50 g dry weight silt-clay coastal sediments. Using 
this method No. 2 fuel oil aromatic hydrocarbons incorporated into marsh 
sediments were precisely measured in samples taken within one week of a spill, 
and eight months after a spill. 

Second: Several sections from a core in Buzzards Bay, Massachusetts have 
been analyzed for alkanes, cycloalkanes, and aromatic hydrocarbons. This is an 
initial attempt at investigating an historical record of anthropogenic fossil fuel 
inputs to coastal sediments. The results indicate an increase of an order of 
magnitude in concentrations of fossil fuel hydrocarbons from circa 1810 to 
1840 to the present. The aromatic hydrocarbon distributions indicate urban air 
hydrocarbons as the major source. 

Third: The input of fossil fuel hydrocarbons from sewage sludge and dredge 
spoils in the New York Bight is discussed. An estimated 3.6 x 10 3 tons of fossil 
fuel hydrocarbons are discharged each year by dumping in this area. 

INTRODUCTION 

Research concerned with chemical pollutants in the environment can be 
most easily divided into two broad areas of investigation: biological effects and 


68 


biogeochemistry. The latter is the category encompassing the three fossil fuel 
pollution investigations summarized in this paper. Each investigation is, or will 
be, the subject of one or more papers, and the reader is referred to these papers 
for details and further discussion. Biogeochemical research delves into the 
sources, distributions, pathways of transfer, reactions, intermittent and 
ultimate fates of pollutants in the environment. 

The incorporation of fossil fuel hydrocarbons into surface sediments as a 
result of oil spills or chronic effluent releases (2, 6, 15), and the resulting 
long-term slow (years) chemical and biochemical removal processes, was a 
major finding of oil pollution research between 1969 and 1974. An important 
concern evolving from these findings was the question of the distribution and 
long-term fate of fossil fuel hydrocarbons in surface sediments. The fossil fuel 
components causing the greatest concern were the aromatic hydrocarbons, 
although recent research has documented that nitrogen containing compounds 
such as p-toluidines and degradation products such as phenalene-l-one, are also 
very toxic to certain marine species (18, 19). Thus, there was a need for 
investigations of aromatic hydrocarbons in surface sediments. This led to a 
search for a means to accurately measure individual aromatic hydrocarbons at 
the 1 to 100 x 10"^ g/g dry weight concentration level in sediments. 
Quantitative gas chromatography-mass spectrometry or mass fragmentography 
has evolved as one of the more discriminating and sensitive methods to apply 
to this problem (10, 11, 13). 

Our quantitative GC-mass fragmentographic method is described in detail in 
another paper (8). We have determined the precision of the method as 2 to 3% 
based on repeated injections of standard aromatic hydrocarbons for 50 x 10"^ 
g and about 12% for 1 x 10'^ g. This compares favorably with quantitative gas 
chromatographic determinations. However, the GC-MF technique has the very 
powerful added advantage of allowing mass spectrum to be scanned to insure 
more complete identification of the compounds measured. A comparison of 
GC-MF determination of the weight percent of selected aromatic hydrocarbons 
in the API reference No. 2 fuel oil with earlier GC measurements (17) is 
presented in Table 6-1. We think that the agreement is quite good. We have 
applied this technique to measuring selected aromatic hydrocarbons in marsh 
sediments exposed to a No. 2 fuel oil spill. 


RESULTS AND DISCUSSION 

Bouchard No. 65 Oil Spill—October, 1974 

On October 12, 1975 the Bouchard Barge No. 65 spilled No. 2 fuel oil into 
Buzzards Bay, Massachusetts. A small amount of this oil entered Windsor Cove 


69 


Table 6-1. Weight Percent of Selected Aromatic 
Hydrocarbons in Fuel Oils. 



Naphthalene 

Naphthalenes 

Ci-Phenanthrenes 

C2‘Phenanthrenes 

API No. 2 Fuel Oil 
(Present Study) 

V 

2.0 ± 0.3 

0.24 ± .02 

0.23 ± .03 

API No. 2 Fuel Oil 
(Warner, Ref. 1 7) 

0.40 

2.7 

0.27 

0.19 

Bouchard Barge No. 65 
No. 2 Fuel Oil Spilled 
October, 1974. 

Buzzards Bay, Mass. 

0.17 ± .01 

0.95 ± .05 

0.37 ± .02 

0.33 ± .04 


(Figure 6-1) and a sheen of oil with accompanying fuel oil odor was present in 
some marsh and intertidal areas of the cove. We selected two sites for a small 
study of the long-term fate of this fuel oil in sediments; a marsh area and an 
intertidal area. The locations of these sites were carefully recorded, and cores 
have been taken every fall in October, and every spring in May or June since 
October, 1974. We did not intend, nor do we pretend, to offer an in-depth 
study of the geographical extent of the spill or long-term fate at several stations 
as was conducted for the West Falmouth oil spill (2, 3, 5). Funding, manpower, 
laboratory space and other commitments to oil pollution research prevented 
such a study. Also, it was our understanding that Commonwealth of 
Massachusetts laboratories were conducting a survey of the geographical 
extent, and long-term fate of the oil. 

Our intent was to compare the long-term fate of the oil at the two locations 
described, with earlier studies of the West Falmouth oil spill. In essence, there 
was a near duplicate experiment in progress. The West Falmouth oil spill 
involved No. 2 fuel oil spilled in late September, 1969 a few miles away from 
where the Bouchard Barge No. 65 spilled No. 2 fuel oil in October, 1974 
(Figure 6-1). Was the West Falmouth oil spill really unique with respect to 
longevity of the spilled oil in marsh and intertidal sediments as some have 
suggested? This was the primary focus of our investigation. The complete set of 
data of our study will be presented elsewhere. We have applied the GC-MF 
technique to a set of marsh cores from October, 1974 and May, 1975. This 
data is presented in Table 6-2. Note that the concentrations of aromatic 
hydrocarbons in the 14-18 or 15-20 cm core section are the concentrations 
present in marsh sediments prior to the spill. The concentrations of aromatic 
hydrocarbons in the surface sediments, 0-6 cm and 0-5 cm, clearly show at 
least two orders of magnitude elevation in concentration as a result of the fuel 
oil spill; and elevated concentrations are still present in May, 1975 although 
they have decreased by a factor of about 5 to 6. The longevity of the aromatic 
hydrocarbons in the marsh sediment is still under investigation. 


70 







41° 42' - 


41° 42' - 


4I°40‘ - 


41° 38' - 


<b 


A 

V 


S 


f 




<6 


CAPE COD CANAL ^ 




BASSETTS I. 


SCRAGGY NECK 


WILD HARBOR 


41 °36‘ - 


CHAPPAQUOIT PT 


41° 34' 


SIPPEWISSET 

J_L 


POCASSET 


WINDSOR COVE 


WEST FALMOUTH 


0 1 
— I - i - 1 - 1 

nouticol miles 


WOOD NECK 


70° 44' 70°42' 70°40' 70°38' 70°36‘ 70°34' 


Figure 6-1. Buzzards Bay, Massachusetts. 


71 







Table 6-2. Concentrations of Naphthalenes/Phenanthrenes in 
Windsor Cove Sediments (^g/gram dry wt. sediment). 


Component 

Oct. 

Depth 

MARSH 

Cone. 

May 

Depth 

Cone. 

Naphthalene 

0-6 cm 

14-18 cm 

9.2 

0.024 

0-5 cm 

1 5-20 cm 

0.63 

0.011 

C^-Naphthalenes 

0-6 cm 

14-18 cm 

370 

1.1 

0-5 cm 

1 5-20 cm 

41 

0.33 

C2'Naphthalenes 

0-6 cm 

14-1 8 cm 

1380 

5.1 

0-5 cm 

1 5-20 cm 

260 

1.5 

Cg-Naphthalenes 

0-6 cm 

14-1 8 cm 

3040 

12 

0-5 cm 

1 5-20 cm 

590 

3.4 

C-j-Phenanthrenes 

0-6 cm 

14-18 cm 

500 

2.2 

0-5 cm 

1 5-20 cm 

97 

0.94 

C2'Phenanthrenes 

0-6 cm 

14-1 8 cm 

480 

2.2 

0-5 cm 

1 5-20 cm 

96 

1.1 


The data in Table 6-2 demonstrate the usefulness of the technique of 
quantitative GC-MF analysis in biogeochemical studies of oil spills. The data 
also demonstrates that fuel oil aromatic hydrocarbons have survived for at least 
seven months in the marsh sediments in concentrations well above background. 
Thus, for this time period, the West Falmouth spill was not unique. Further 
investigation will determine if the parallel between the fate of the petroleum 
compounds from the two oil spills will continue. 

Historical Record of Fossil Fuel Hydrocarbons 
in Buzzards Bay, Massachusetts 

Our gas chromatographic measurements of hydrocarbons in surface 
sediments at several locations in Buzzards Bay, Massachusetts indicated the 
presence of an unresolved complex mixture of hydrocarbons with a wide 
molecular weight range, indicating that these hydrocarbons might be from 
chronic oil pollution (8). However, there were several other sources such as 
natural diagenetic processes and weathering of ancient sediments to be 
considered (8). In order to assist in evaluating the source of these 


72 





hydrocarbons, we obtained sediment cores at several locations in coastal areas 
of the western North Atlantic (8). At Station P Pb-210, geochronology 
measurements were obtained (7). These measurements and the measurements 
of Pu-239/240 and Cs-137 at this same location by others (4) allowed us to 
estimate sedimentation rates. We then measured hydrocarbon concentrations in 
several core sections at Station P. We also applied quantitative GC-MF analyses 
to measure phenanthrene and Cj and C-> phenanthrenes. The results of these 
measurements, as reported in (8), are given in Table 6-3. It is clear that circa 
1900 concentrations of hydrocarbons constituting the unresolved complex 
mixture increased, as did the concentrations of phenanthrenes. The ratios of 
the C| and C 0 phenanthrenes are not those found in spilled oil. Instead, the 
ratios indicate that these aromatic hydrocarbons are from pyrolytic sources 
(13, 20). Our hypothesis is that these hydrocarbons are primarily from direct 
and remobilized urban air hydrocarbons (8). We have determined that there is a 
trend of decreasing concentrations of UCM hydrocarbons with increasing depth 
in a core at another station in Buzzards Bay (10), and a station in the Gulf of 
Maine (Figure 6-2). 

Furthermore, similar results have been reported for Lake Washington, 
Seattle, Washington sediments (16). A much more detailed analysis of the 
aromatic hydrocarbons in three sections of another core from Station P, 


Table 6-3. Hydrocarbons and Chlorinated Hydrocarbons 

in Station P Core Sections 


Section 

Average Time 
of Deposition 

UCM a 

(^g/g) 

Phenanthrenes 

(ng/g) 

C Q C-, 

c 2 

0-1 cm 


74 

NA b 

44 

38 

1-2 cm 


105 

34 

27 

28 

8-12 cm 

1940 

44 

NA 

31 

29 

20-24 cm 

1900 

12 

15 

11 

10 

54-58 cm 

1790 

5.2 

3.7 

2.9 

2.8 

58-62 cm 

1780 

6.2 

NA 

NA 

NA 


a Mixture of alkanes and cycloalkanes - indicates petroleum hydrocarbons. 

L 

°NA — not analyzed. 


73 








Figure 6-2. Gulf of Maine. 


74 





Buzzards Bay, have recently been completed (12). These analyses, also by 
GC-MF, greatly extend the earlier analyses for the Buzzards Bay station, and 
establish that polynuclear aromatic hydrocarbons (PAH) from a combustion 
source increase by at least an order of magnitude in sediment deposited after 
about 1850. Another recent paper (14), has reported a detailed study of PAH 
in a core from Lake Constance in the Federal Republic of Germany. The 
surface sediments of this core contained increases of PAH concentration of 50 
to 100 times that of sediments deposited circa 1800. The PAH composition 
again indicated a pyrolytic source. 

The implications of these findings are that coastal and lacustrine 
environments, especially the benthic ecosystems, have been exposed to 
increased PAH concentrations over the past several decades. Whether or not 
this chronic long-term increase in PAH concentration reflects a substantial 
environmental risk is not known, and a detailed discussion is beyond the scope 
of this paper. It is important, though, to consider that many of the PAHs are 
known to have adverse effects on marine organisms (13). The benthic 
ecosystems may have been “stressed” by PAH pollution for some time. This is 
an important point to keep in mind when considering control stations for 
studying oil spills in coastal areas. For example, the control stations for the 
studies of the effects of the West Falmouth oil spill on subtidal benthos were 
not very far from the two stations, P and D, we have sampled in Buzzards Bay. 
Does this mean that these stations are truly “normal” with respect to the 
effects of aromatic hydrocarbons, or have they also been subtly, chronically 
affected by the increasing amounts of PAH deposited from direct and 
remobilized urban air PAH? 

New York Bight Surface Sediments 

We have estimated the rate of fossil fuel hydrocarbons discharged by 
dumping in the New York Bight is about 3.6 x 10^ tons/year (9), or about 2% 
of the estimated global discharge of 180 x 1(P tons per year of petroleum 
hydrocarbons from routine operations and spills associated with outer 
continental shelf drilling and production (3). The composition of PAH in the 
New York Bight dump site surface sediments indicates that these hydrocarbons 
are primarily of pyrolytic origin (13). The fossil fuel hydrocarbons most likely 
are from urban air fallout, and are swept into storm sewers and municipal 
sewers by rain water, and are either discharged to New York harbor or become 
associated with the sewage sludge in the treatment plants. Dredge spoils from 
the harbor and sewage sludge are then dumped in the New York Bight resulting 
in delivery of PAH and other pollutants to the continental shelf area. Since 
there are other dump sites off the East coast of the U.S., the input of 
petroleum hydrocarbons from this source must be larger than in the New York 
Bight alone. Thus, significant and measurable quantities of contaminant 


75 


hydrocarbons are already being deposited in continental shelf areas off the 
eastern United States before Outer Continental Shelf oil and gas drilling and 
production have begun. This must be taken into account when assessing 
potential environmental impacts of OCS operations, now and in the future. 

SUMMARY AND GENERAL DISCUSSION 

Aromatic hydrocarbons are incorporated into surface sediments as a result 
of oil spills, and the chronic dribbling of urban air hydrocarbons into the 
marine environment. These compounds are known to have adverse effects on 
marine organisms under certain conditions. The challenge posed is to conduct 
experiments which will investigate how bottom current resuspension, 
bioturbation by animals, and long-term microbial and chemical processes act 
individually and collectively on the aromatic hydrocarbons in surface 
sediments. Are these compounds in the sediments incorporated into benthic 
organisms? At what rate and under what conditions? We need to relate 
chemical analyses by some means to biological availability. 

Some recent investigations conducted on a short-term two-week exposure of 
sipunculid worms suggest that naphthalenes can be ingested from naphthalene 
contaminated sediments (1). Two weeks of “depuration” in a clean 
environment removed all measurable quantities of naphthalenes from the 
worms (1). The exposure time was very short. What happens when exposure of 
the benthic organism is continuous for years, as is probably the case for low 
concentrations of polynuclear aromatic hydrocarbons in Buzzards Bay, and 
higher concentrations near the New York Bight area? 

ACKNOWLEDGMENTS 

I wish to thank R.A. Hites, R.E. LaFlamme, B.W. Tripp, N.M. Frew and J.M. 
Teal for enjoyable collaboration and discussion of much of the research 
discussed and referenced in this paper. This paper presents a synopsis of several 
papers and acknowledgments to agencies providing financial support are found 
in the referenced papers. 

The compilation of data and writing of this paper were supported by U.S. 
Environmental Protection Agency Grant R803902. This paper is Contribution 
Number 4041 of the Woods Hole Oceanographic Institution. 

REFERENCES 

1. Anderson, J.W., L.J. Moore, J.W. Blaylock, D.L. Woodruff and S.L. 
Kiesser. 1977. Bioavailability of Sediment-Sorbed Naphthalenes to the 
Sipunculid Worm, Phascolosoma agassizzii. Chapter 29 in Fate and Effects 


76 


of Petroleum Hydrocarbons in Marine Organisms and Ecosystems, Wolfe, 
D.A. (ed.), Pergamon Press, New York. 

2. Blumer, M. and J. Sass. 1972. Oil Pollution: Persistence and Degradation of 
Spilled Fuel Oil. Science 176:1120. 

3. Blumer, M. and J. Sass. 1972. The West Falmouth Oil Spill, Data Available 
1977. II. Chemistry Technical Report No. 72-19, Woods Hole 
Oceanographic Institution, unpublished manuscript. 

4. Bowen, V.T. 1975. Transuranic Elements in Marine Environments. In: U.S. 
Energy Research and Development Administration Health and Safety 
Laboratory Report HASL-291 April 1, 1975, pp. 157-179. 

5. Burns, K.A. and J.M. Teal. 1971. Hydrocarbon Incorporation into the Salt 
Marsh Ecosystem from the West Falmouth Oil Spill. Technical Report No. 
71-69, Woods Hole Oceanographic Institution, unpublished manuscript. 

6. Farrington, J.W. and J.G. Quinn. 1973. Petroleum Hydrocarbons in 
Narragansett Bay. I. Survey of hydrocarbons in Sediments and Clams 
(Mercenaria mercenaria). Estuarine and Coastal Marine Science 1:71. 

7. Farrington, J.W., S.M. Henrichs and R. Anderson. 1977. Fatty Acids and 
Pb-210 Geochronology of a Sediment Core from Buzzards Bay, 
Massachusetts. Geochemica et Cosmochimica Acta 41:289. 

8. Farrington, J.W., N.M. Frew, P.M. Gschwend and B.W. Tripp. 1977. 
Hydrocarbons in Cores of Northwestern Atlantic Coastal and Continental 
Margin Sediments. Estuarine and Coastal Marine Science (in press). 

9. Farrington, J.W. and B.W. Tripp. 1977. Hydrocarbons in Surface Sediments 
of the Western North Atlantic. Geochimica et Cosmochimica Acta (in 
press). 

10. Frew, N.M. and J.W. Farrington. 1977. Mass Fragmentographic 
Determination of Aromatic Hydrocarbons in Marsh and Coastal Sediments. 
Submitted to Environmental Science and Technology. 

11. Hase, A., P.H. Lin and R.A. Hites. 1976. Analysis of Complex Polynuclear 
Aromatic Mixtures by Computerized GC/MS. In: Carcinogenesis, Jones, 
P.W. and Freudenthal, R.I. (eds.), Raven Press, N.Y., Vol. I. 

12. Hites, R.A., R. LaFlamme and J.W. Farrington. 1977. Polycyclic Aromatic 
Hydrocarbons in Recent Sediments: The Historic Record. Science (in 
press). 


77 


13. LaFlamme, R.E. and R.A. Hites. 1977. The Global Distribution of 
Polycyclic Aromatic Hydrocarbons in Recent Sediments. Geochimica et 
Cosmochimica Acta (in press). 

14. Miller, G., G. Grimmer and H. Bohnke. 1977. Sedimentary Record of 
Heavy Metals and Polycyclic Aromatic Hydrocarbons in Lake Constance. 
Die Naturwissenshaften (in press). 

15. National Academy of Sciences. 1975. Petroleum in the Marine 
Environment. Washington, D.C. 

16. Wakeham, S.G. and R. Carpenter. 1976. Aliphatic Hydrocarbons in 
Sediments of Lake Washington. Limnology and Oceanography 21:711. 

17. Warner, J.S. Battelle Memorial Institute. Unpublished data. 

18. Winters, K., R. O’Donnel, J.C. Batterton and C. Van Baalen. 1976. 
Water-soluble Components of Four Fuel Oils: Chemical Characterization 
and Effects on Growth of Microalgae. Marine Biology 36:269. 

19. Winters, K., J.C. Batterton and C. Van Baalen. 1977. Phenalene-l-one: 
Occurrence in a Fuel Oil and Toxicity to Microalgae. Marine Biology (in 
press). 

20. Youngblood, W.W. and M. Blumer. 1975. Polycyclic Aromatic 
Hydrocarbons in the Environment: Homologous Series in Soils and Recent 
Marine Sediments. Geochimica et Cosmochimica Acta 39:1303. 


78 


IDENTIFICATION OF ENVIRONMENTAL 
GENETIC TOXICANTS 
WITH CULTURED MAMMALIAN CELLS 


Alexander R. Malcolm, Robert R. Young, 
and Carolyn J. Wilcox 


U.S. Environmental Protection Agency 
Environmental Research Laboratory 
Narragansett, R.l. 02892 


ABSTRACT 

Experiments designed to detect small-scale mutations leading to auxotrophy 
were carried out in vitro with the Chinese hamster ovarian (CHO) cell system 
(5-bromodeoxyuridine/visible light selection) initially described by Puck and 
Kao (43). The system was standardized with ethylmethanesulfonate (EMS), a 
known mutagen previously demonstrated to be active in CHO cells (27), and 
5-bromodeoxyuridine (BrdU), another known mutagen (7) utilized in the 
selection procedure, but not previously evaluated for mutagenic activity in the 
CHO Cell/BrdU-VL assay. Both EMS and BrdU routinely yielded glycine, 
hypoxanthine or triple-requiring (glycine/hypoxanthine/thymidine) 
auxotrophs and showed dose response. For a series of inorganic compounds 
known to be or suspected of being genetic toxicants, statistically significant 
numbers of auxotrophs were obtained only with the chloride salts of cadmium 
and manganese. Neither cadmium nor maganese were consistently mutagenic, 
cadmium showing activity in about 20 percent of experiments, manganese in 
50 percent of experiments. It was not possible to demonstrate dose response 
with these compounds. A water extract of JP-5 jet fuel was also found to be 
mutagenic in a single test. Variant cell types other than auxotrophs were 
isolated from cell populations treated with three different carcinogenic agents 
(EMS, CrO^, PbAc 0 .3H->0) but not from control experiments. These cells, 
exhibiting either a rounded cell morphology or potential contact inhibition, 
may reflect mutation in additional loci of possible value as genetic markers. 
Other data are presented to illustrate special problems associated with the 
application of in vitro cell systems. 

INTRODUCTION 

Serious concern for the possible effects of genetic toxicants in the 
environment developed approximately a decade ago with the discovery of 


79 


chemical mutagens capable of inducing high frequencies of mutation at high 
levels of survival (17). Concern also stemmed from the realization that man was 
greatly expanding the number of compounds theoretically capable of 
increasing mutation frequencies beyond present ‘spontaneous levels. As 
recently stated at an open meeting sponsored by the United States Department 
of Health, Education and Welfare on the value of selected test systems to 
detect and assess the mutagenic activity of chemicals (21), a human disease 
burden exists which is of genetic origin. Increases in mutation frequency can be 
expected to enlarge this burden, and many classes of chemicals already in the 
environment are known to include genetic toxicants. Although uncertainty 
remains regarding the precise impact such compounds might have upon human 
health, there is justification for apprehension (15). 

Genetic toxicology, a new branch of toxicology concerned with the 
identification and evaluation of DNA-damaging agents (carcinogens, mutagens 
and some teratogens), may be broadly divided into (a) screening tests for 
identification of potential toxicants, (b) procedures for estimating risk, and (c) 
techniques for population monitoring. Screening involves primarily the use of 
rapid, inexpensive assays which detect agents capable of damaging or altering 
DNA. Because DNA is chemically and structurally similar in most organisms, 
and is considered the probable target of genetic toxicants, any organism or 
appropriate part thereof may be theoretically employed as a screening tool. 
Accordingly, viruses (18), a variety of microbial systems (3,36), cultured 
animal cells (14, 39, 26), Drosophila (2, 48), and various subcellular assays 
designed to measure effects directly on DNA (47, 49) are widely used for 
screening purposes. Several short-term tests utilizing intact mammals are also 
available for screening (31). 

This paper is concerned with the application of an in vitro mammalian cell 
assay utilizing nutritional markers as an indicator system for genetic toxicants 
detected as mutagens. Major objectives are to (a) outline techniques for 
measuring the acute toxicity of chemicals to cultured cells, (b) qualitatively 
describe the CHO Cell/BrdU-VL system as an assay for mutation, (c) present 
data relative to the mutagenic potential of a series of compounds known to 
accumulate in the tissues of edible marine organisms (41,45, 52) or which have 
been associated with the occurance of neoplasias in such organisms (8, 51), and 
(d) illustrate some additional end points, as well as some potential problems, 
pertinent to the application of in vitro cell assays. A detailed description of 
equipment, reagents, special techniques and experimental procedures relevant 
to the CHO Cell/BrdU-VL system will not be given here. A general protocol for 
this assay has been published by Kao and Puck (28). Our modifications to their 
procedure will be described elsewhere (33). 


80 


METHODS 


Acute toxicity 

Before compounds can be evaluated for mutagenic activity, their acute, 
physiological toxicity must be determined. This is accomplished by measuring 
the ability of single cells to produce macroscopic colonies arising in 
experimental dishes following exposure to specific concentrations of the test 
agent for specified periods of time. Relative plating efficiency (RPE), defined 
as the ratio of macroscopic colonies arising in experimental dishes, to those 
appearing in controls, may be plotted against dose to yield survival curves of 
the type shown in Figure 7-1. Concentrations of compound to be tested for 
genetic activity are selected from such curves. The exponential portion of each 
curve is described by equation [1] where (S/S ) is the surviving cell fraction or 
percent RPE, (n) is the hit or target number (30) and (D/D Q ) is relative dose 
(44). The target number is defined operationally by the intersection of the 
exponential portion of the curve with the ordinate axis when the former is 
extrapolated back. Relative dose is defined as the ratio of the experimental 
molar concentration of toxicant (D) to that increased in molar concentration 
(D q ) required to reduce the cell population by the fraction (1/e). The value of 
D q is, for each compound, obtained from a plot of molar concentration versus 
surviving cell fraction. Because chemicals differ in their molar toxicity by 
orders of magnitude, relative dose provides a convenient way to depict survival 
data for many compounds simultaneously. It is noted that the random hit 
model expressed by equation [1] was derived for radiation effects (30), and 
requires interpretive modifications when describing cell inactivation by 
chemicals (32). The use of plating efficiency to asses acute chemical toxicity 
has been described elsewhere (34). 

The CHO Cell/BrdU-VL System 

Figure 7-2 represents a simplified and generalized protocol for inducing, 
isolating and characterizing mutant cells. Initially, cells are inoculated into 
dishes or flasks and allowed to attach to the plastic substratum. Following 
attachment, cells are exposed to the test agent at one or more concentrations. 
The cells are then washed free of the test compound and fresh medium added. 
During the expression period, cells are grown under nonselective conditions, 
permitting induced genetic damage to become fixed into DNA, and ultimately 
to become expressed at the cellular level. The length of the expression period is 
a function of the system employed, the genetic markers involved, and the 
conditions of the experiment. Selection represents the application of a set of 
conditions permitting mutant cells to survive while eliminating wild-type cells. 
Selective conditions employed are specific for the type of mutant sought. Once 
potential mutants have been isolated, they may be subjected to genetic analysis 
for confirmatory purposes and for further characterization. 


81 


o 

oo 

X 

CD 

'rO 



NO IlDVdd 1130 ON IAI AdflS 


82 


RELATIVE DOSE 

Figure 7-1. Typical 16-hour survival curves for five test compounds. 

Note: The curves are constructed from relative plating efficiency data by plotting the surviving cell fraction (S/S Q ) against relative 
dose (D/D 0 ). Concentrations or doses of toxicant employed in mutagenesis experiments are selected from such curves. 





GENERALIZED AND SIMPLIFIED PROTOCOL FOR POINT MUTATION IN CELLULAR SYSTEMS 


CELL INOCULATION 
AND 

EXPOSURE TO 
TEST AGENT 

MUTANT 

EXPRESSION 

MUTANT CELL 
SELECTION 

MUTANT CELL 
ANALYSIS 

ATTACHMENT 






WASH 


Figure 7-2. A generalized and simplified protocol for the 
induction, isolation and characterization of mutations 

in cellular systems. 


Figure 7-3 represents a protocol containing the same basic features as that in 
Figure 7-1, but is specific for the CHO Cell/BrdU-VL system. In this technique, 
CHO-K1 cells are cultured in two types of media. One medium (FI2D) 
contains the minimal nutritional requirements for the growth of single cells 
into macroscopic colonies with high efficiency. A second, enriched medium 
(FI2) is constructed from the minimal by addition of nine nutrients (alanine, 
glycine, aspartic acid, glutamic acid, lipoic acid, vitamin B^, inositol, 

A B C 



chemical 

treatment 


mutation 

induction 





mutant cell 
selection 


5 -bromodeoxyuridine 



exposure 

to 

white 

light 


colony of 
mutant = 
cells 



mutant cell growth 
<- 





E 


D 


Figure 7-3. Schematic representation of the Chinese hamster 
ovarian cell system (BrdU-visible light selection procedure) for 
the detection of small-scale mutations. 


Note: (A) Population of wild-type cells. (B) Nutritional mutant (auxotroph) 
following induction and expression. (C) Wild-type cells with BrdU-containing 
DNA. (D) Surviving mutant cell following selective elimination of wild-types 
via the combined action of BrdU and white light. (E) Colony of mutant cells 
which grew in enriched medium. (After Kao and Puck (28)). 


83 











thymidine and hypoxanthine) not required exogenously by the cells for 
optimal growth. Mutants are detected by screening populations of cells 
exposed to test compounds for nutritionally deficient forms (auxotrophs) 
requiring one or more of the nine nutrilites omitted from FI 2D medium. 

Operationally, lO^ 7 cells are exposed in four parallel cultures to single or 
multiple doses of the test agent, following inoculation and cell attachment 
(point A, Figure 7-3). Because even induced mutation is a rare event, there will 
generally be, following expression, a small number of mutant cells growning 
among millions of nonmutants in enriched medium. To identify the mutants, it 
is necessary to introduce a procedure which will eliminate the prototrophic 
(wild-type) cells, while allowing auxotrophs to survive. This is accomplished by 
taking advantage of the fact that mutants auxotrophic for one or more of the 
nine nutrilites omitted from FI 2D medium will be unable to grow in this 
medium, whereas wild-types will. Thus, at point B, Figure 7-3, FI2 medium is 
replaced with FI 2D medium. This initiates the selective process by effectively 
terminating protein and nucleic acid synthesis. The thymidine analog, BrdU, is 
then added to the FI 2D medium from which it is incorporated into the DNA 
of wild-type cells (point C, Figure 7-3). Subsequent illumination of the cell 
population with white light is lethal to those cells having incorporated 
sufficient BrdU. Mutant cells do not incorporate BrdU, and survive the 
selective process (point D, Figure 7-3). Wild-type cells survive selection to an 
extent approaching 0.02 percent. In the presence of FI2 medium, mutant cells, 
along with some wild-types, grow into macroscopic colonies (point E, Figure 
7-3). These are picked and tested for mutant identification. It is important to 
note that the use of a known mutagenic agent (BrdU) in the selective process is 
of no consequence, as the only cells incorporating BrdU are wild-types destined 
for death. Mutants to be isolated are existent in the population at the end of 
the expression period prior to the application of selective conditions. 

The procedure illustrated in Figure 7-3 and described above was applied in 
collecting the mutagenesis data presented below. Figure 7-4 shows the lighting 
apparatus used to illuminate cell populations following exposure to BrdU. 
Figure 7-5 shows cell survival in four randomly selected dishes several days 
after illumination. Cells not killed in selection have grown into macroscopic 
colonies. Because mutant and wild-type cells differ only in their requirements 
tor exogenous nutrilites, they may be distinguished only by analysis of their 
growth properties in enriched and deficient media. 

Statistical Analysis of Data 


When testing compounds for mutagenic activity, we usually want to know if 
the number of mutants per unit number of viable cells screened is sufficiently 
larger in experimental, versus control situations, to support a conclusion of 


84 



Figure 7-4. Illumination apparatus for the inactivation of cells 

with BrdU-containing DNA. 

Note: Plastic culture dishes containing 2x10^ cells each are subjected to 


white fluorescent light for 60 minutes. 


Figure 7-5. Macroscopic colonies in four randomly selected dishes 
approximately seven days after illumination with white light. 

Note: These clones, originating from cells surviving selection, are tested to 
determine if any are auxotrophic mutants. 


85 













induced mutation by the test compound. In the CHO Cell/BrdU-VL assay, each 
cell screened will either be auxotrophic for one or more of the nine nutrilites 
omitted for FI 2D (operational definition of mutant), or it will not be 
(operational definition of wild-type). The actual criteria employed to classify 
mammalian cell variants as true mutants are somewhat complex, and have been 
reviewed recently (16, 40, 46). For the purpose of this paper, the operational 
definitions given above shall be used. 

In the standard procedure, a sample of 10 1 cells from each test or control 
population is distributed among fifty 60 mm dishes, and subjected to the 
selective process. Surviving clones are then sampled and tested for the presence 
of auxotrophs. The total number of auxotrophs expected per 10^ viable cells is 
then estimated from the data by equation [2], where (y) is the estimated 
number of auxotrophs, (x) is the number of auxotrophs observed, (n) is the 
number of replica experiments, (A) is the total number of cells surviving 
selection, (B) is the total number of colonies picked and tested, (C) is the 
initial number of 60 mm dishes, (D) is the final number of dishes (some may be 
lost to contamination during the course of the experiment), and (E) is the 
absolute plating efficiency (defined as the ratio of macroscopic colonies 
produced to cells inoculated) as measured in low density control dishes. 
Because mutants are randomly distributed among wild-types in mixed 
populations, the probability that any given survivor will be a mutant should be 
constant over all survivors. Moreover, as only a small number of mutants is 
generally found in any given population, the distribution of mutants in such 
populations should be Poisson. Accordingly, mutagenesis data from sets of 
replica experiments were tested for goodness of fit to a Poisson model, and 
found to be consistent with this type of distribution (33). 


For two independent Poisson variables (X, Y), a new statistic (V) has been 
proposed by Best (9) for testing the difference between two Poisson 
expectations (e.g., the estimated mean number of mutants in experimental (X) 
versus control (Y) populations). This statistic, given by equation [3], is similar 
in performance to the more familiar square root of the Poisson Index of 
Dispersion (20), except in the tails of the distribution where (V) is superior. 
Although (V) is a function of the Poisson variables (X, Y), (V) itself shows an 
approximately normal distribution. This statistic may be particularly applicable 
to mutagenesis data where the difference in variance observed between 
experimental and control populations is large. This is the situation at the 
present time with the CHO Cell/BrdU-VL system where mutants are rarely 
observed in control populations. All mutagenesis data considered below were 
scaled via equation [2] and compared to an historical control (Y) in 
accordance with equation [3] and appropriate confidence limits. The model 
given by equation [3] and appropriate confidence limits. The model given by 


86 


equation [3] is at present a proposed one and may not be the final model of 


choice. 



(y) 

= (x/n) [(A)/(B)] [(C)/(D)(E)] 

[2] 

(V) 

= (2X + 3/4)1/2 . (2y + 3/4)1/2 

[3] 

RESULTS 




Control Investigations 

With the exception of the spontaneous proline auxotroph isolated as the K1 
subclone of the CHO cell (44), other spontaneous auxotrophs with 
requirements for one or more of the nutrilites omitted from FI 2D medium had 
not been previously reported for the CHO Cell/BrdU-VL system. Such 
auxotrophs could easily be suppressed in stock cell populations by maintaining 
cells in minimal rather than enriched medium. This was not done in order to 
determine if spontaneously arising auxotrophs could indeed be identified in 
control or stock populations. Table 7-1 summarizes data from 16 different 
control experiments carried out over a period of several months. Two glycine 
mutants were identified among 989 clones picked and tested. The observed 
frequency of spontaneous auxotrophy is thus two mutants per 1.2 x 10^ viable 
cells. These data, in combination with data for the other parameters of 
equation [2], were utilized to obtain an estimate of 0.331 mutants per 10^ 
viable control cells. This value was substituted for (Y) in equation [3]. 


Table 7-1. Summary Mutagenesis Data From Several 

Control Experiments 


(n) Viable [(A)/(B)] [(C)/(D)(E)] Auxotrophys isolated (y) 

cells Gly Hyp (x) 

16 1.2 x 10 7 1888/989 (800)/(770) (.750) 2 0 2 0.331 


The scaling of (x) by equation [2] does not consider the fact that mutant 
cells may be lost to the effects of starvation during selection. In fact, 
reconstruction experiments employing known numbers of mutants have 
demonstrated that this type of loss does occur for the three types of 
auxotrophs observed (27, 33). Consequently, equation [2] underestimates 
actual mutant frequencies. 


87 





Induced Mutation with Standard Mutagens 


The standard mutagens, EMS and BrdU, were evaluated for mutagenic 
activity at several doses. Data pertinent to the mutagenicity of these 
compounds are presented in terms of the parameters of equation [2] in Table 
7-2. The estimated number of mutants per 10 6 viable cells (7) is plotted in 
Figure 7-6 as a function of relative dose. By expressing the mutagenesis data in 
terms of mutant cell frequencies per 10^ viable cells, meaningful comparisons 
between different compounds or different doses of the same compound could 
be made. Applying the data to equation [3], EMS was mutagenic at all doses 
tested (a< 0.05). BrdU was mutagenic at five of seven doses tested, and 
produced a complex dose-response pattern similar to those observed with 
hycanthone methanesulfonate, and other compounds in different assays (12, 
13 ). 

Induced Mutation with Other Compounds 

Forward mutation experiments employing doses of toxicant generally 
yielding 20 percent survival or greater were carried out in replica with several 
inorganic compounds, and an aqueous extract of JP-5 jet fuel. The data are 
presented in Table 7-3 in terms of the parameters of equation [2]. These 
compounds or mixtures were selected for evaluation as mutagens because they 
were either known to have, or were suspected of having, carcinogenic 
properties. Their mutagenic response in the CHO Cell/BrdU-VL system can be 
divided into three classes: (1) Experiments with the oxides of arsenic and 
selenium, lead acetate, and the chloride salts of cobalt and nickel, failed to 
produce any auxotrophs; (2) tests with beryllium and chromium usually 
produced auxotrophs, but never in sufficient numbers to support a conclusion 
of induced mutation; (3) Experiments with cadmium chloride, manganese 
chloride, and an aqueous extract of JP-5 jet fuel, also produce auxotrophs, 
sometimes in sufficient numbers to suggest induced mutation by these 
compounds. As indicated in Table 7-3, it was possible to obtain relatively large 
numbers of auxotrophs with CdCl->. Usually, however, observed mutant 
frequencies were low. Cadmium chloride was found to be significantly 
mutagenic in about 20 percent of experiments, as was the extract of JP-5 jet 
fuel. Manganous chloride was observed to be mutagenic in approximately 50 
percent of experiments. 

Isolation of Nonauxotrophic Variants 

Two classes of variants, other than auxotrophs, were isolated from cell 
populations treated with known genetic toxicants. One class consists of cells 
exhibiting a rounded morphology, and represents cells unable to stretch out on 


88 


Table 7-2. Data From Forward Mutation Experiments With EMS and BrdU 


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6 (BrdU) 4.60 x 10 H 1 2.8x10° 214/128 (050(/(050)(0.280) 1 4 5 30.1 

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11.63 

Denotes significance at the 95% confidence level; ** denotes significance at the 99% confidence level. 





lOCh 



RELATIVE DOSE 

Figure 7-6. Mutation frequency induced by EMS and BrdU 

as a function of dose. 


the plastic substratum and assume the standard epithelial morphology (Figure 
7-7). Although this type of cell produces colonies of sufficient size to permit 
cloning, the cells are continuously in a rounded state, as if entering mitosis. 
When grown in medium containing 10 percent fetal calf serum, some of the 
clones assume a more normal morphology, and may represent mutants with 
increased serum requirements (27). To date, this type of variant has been 
observed only in populations treated with EMS, BrdU, and compounds of 
chromium, cadmium and lead. 

A second type of variant, appearing to possess the property of contact 
inhibition (1, 19), was isolated from cell populations treated with the known 
carcinogens, EMS, CrO^ and PbAc2*3H-»0. These were detected as wild-type 
cells surviving selection, and which possessed a pronounced fibroblastic 
morphology (Figure 7-7). When cells were grown into confluent monolayers, 
unlike the transformed CHO-K1 cell, they ceased to divide and assumed a state 
of contact inhibition, or a state resembling that of contact inhibition. Dense 
monolayers remained for as long as two weeks without medium changes, and 
without significant deterioration. Confluent sheets of cells could be easily 
trypsinized and dispersed into uniform, single-cell populations. Upon replating 
in fresh growth medium, cells grew with a generation time of approximately 14 
hours, ceasing to divide when the monolayer again became confluent. 


90 




Table 7-3. Data From Forward Mutation Experiments with Selected Additional Compounds 


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Denotes significance at the 95% confidence level; ** denotes significance at the 99% confidence level. 






Figure 7-7. Epithelial and Fibroblastic Morphologies Exhibited by 

the CHO-K1 Cell. 


Note: (A) shows the standard epithelial morphology usually adopted by the 
cell. (B) illustrates the highly fibroblastic form assumed by a potentially 
contact-inhibited cell isolated from a population treated with the known 
carcinogenic agent chromium trioxide. 


92 






Preliminary experiments suggest that these cells show contact inhibition of 
replication in colonies containing at least 100 cells, as well as in dense 
monolayers (35). All such clones tested to date appear stable in their ability to 
express the apparent contact-inhibited state. 

Mutation Frequency and Expression Time 

Mutation is a complex biological process involving much more than 
interaction of mutagen with DNA, or more generally, with the DNA-replicating 
system. The mutagen-DNA interaction usually results not in mutation, but in 
the creation of premutational lesions which become expressed as mutations 
only after a series of additional events has occured (6). For example, 
auxotrophic mutants usaully become expressed as soon as the cells become 
depleted of normal gene product. The length of time required for this and 
other preliminary events to occur may vary with the nature of the gene 
product, the specific mutagen utilized, and the doses employed (5, 7). To 
determine the influence of expression time on observed mutation frequency 
for the mutant phenotypes reported above, experiments employing variable 
expression time were carried out with EMS and BrdU. Doses used were those 
giving maximal observed mutation frequency when the expression time was 

Five days (3 x 10"^ molar for EMS, 1 x lcH - for BrdU). Expression time was 
varied between two and eleven days. The data are given in Table 74, again in 
terms of the parameters of equation [2]. Observed mutation frequency is 
plotted in Figure 7-8 as a function of expression time. 

Optimal expression time is defined as the interval between mutagen 
quenching, and the application of selective conditions yielding maximal 
mutation frequency when dose is held constant. This is observed to be five 
days for BrdU and eight days for EMS. Moreover, for a two-day expression 
period, statistically significant numbers of mutants could always be identified 
in populations exposed to EMS, whereas none are found in populations treated 
with BrdU. Once optimal expression time is exceeded, mutant cell frequency 
decreased rapidly, suggesting that these types of mutants are at a replicative 
disadvantage relative to wild-type cells under nonselective conditions. The fact 
that optimal expression time was different for the two mutagens when tested 
at doses showing similar toxicity, suggest that expression time may be mutagen 
dependent. This leads support to the contention that assays utilizing variable 
expression time shall be required when screening compounds for mutagenic 
activity (4). This may be particularly important for weak mutagens. 

DISCUSSION 

The genetic toxicology of inorganic compounds has largely been ignored, 
despite the fact that many are ubiquitous, highly toxic, and implicated as 


93 


Table 7-4. Auxotroph Frequency as a Function of Expression Time for Single Doses of EM 


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human carcinogens. Neglect of the inorganics in this regard seems due, in part, 
to the fact that such compounds comprise a relatively small percentage of 
known or potential genetic toxicants, are difficult to handle in many 
experimental situations, appear to mediate genetic effects by obscure 
mechanisms, and have not been active in many major assays. Some metals have 
shown activity in DNA repair tests employing microbial systems (37), and in 
reverse mutation assays with E. coli (50). Recently, selenate and some 
compounds of chromium have been shown to be active in selected strains of 
Salmonella typhimurium (38). Perhaps the best assay developed to date for 
predicting the potential genetic toxicity of metals is the in vitro 
DNA-synthesizing system described by Sirover and Loeb, and which detects 
copying errors in replicating DNA (47). The fact that the carcinogenic metals 
were not generally active in the CHO Cell/BrdU-VL system may reflect a 
sensitivity problem related to the loss of mutants during selection to the effects 
of starvation. Because mutation is periodically observed with certain of these 
compounds, factors other than assay sensitivity may be involved. These factors 
could be uniquely important to the expression of mutagenic activity by metals 
and may not be presently under control or consideration. Extensive testing 
with cadmium chloride and beryllium chloride indicates that the problem does 
not lie with expression time. 


95 




The isolation of potentially contact-inhibited cells from populations treated 
with known carcinogenic agents is interesting for several reasons. Among the 
more intriging ideas is the possibility that such cells represent back 
transformation to the noncancerous state. The CHO cell possesses many of the 
properties of transformed cells, including the loss of contact inhibition (42, 
44). In accordance with the somatic cell mutation hypothesis for cancer (10, 
22), agents inducing cell transformation through mutation should, in at least 
some cases, be capable of inducing back transformation in individual cells via 
true reverse mutation or via forward mutation at suppressor loci. Indeed, 
spontaneous revertants of cells transformed to the malignant state in vitro by 
viruses and chemicals have already been described (23, 24). Reversible 
conversion of transformed cells to the contact-inhibited state has been 
observed during exposure of such cells to dybutryl cyclic AMP (25) or 
concanavalin A (11). It remains to be shown if the same agents capable of 
inducing transformation form normal to malignant state can also reverse it, 
such that the revertants are stable in the absence of inducing agent. Although 
virtually all cell-transforming agents tested to date are also mutagens, it is not 
yet possible to say if the phenomenon of cell transformation involves mutation 
(29). Investigation of the properties of these cells is continuing. 

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128-142. 

2. Abrahamson, S., and E.B. Lewis. 1971. The Dectection of Mutagens in 
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3. Ames, B.N., et al. 1973. Carcinogens are Mutagens: A Simple Test 
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5. Arlett, C.F., and S.A. Harcourt. 1972. Expression Time and Spontaneous 
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96 


7. Auerbach, C. 1976. Mutation Research. Halsted Press. London. 

8. Barry, M., P.P. Yevich, and N.H. Thayer. 1971. Atypical Hyperplasia in the 
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10. Bovari, T. 1929. The Origin of Malignant Tumors. Williams and Wilkins. 
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11. Burger, M.M., and K.D. Noonan. 1970. Restoration of Normal Growth by 
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12. Clive, D. 1973. Mutagenicity of Hycanthone and Several of Its Analogs in 
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13. Clive, D. 1974. Mutagenicity of Thioxanthines (Hycanthone, Lucanthone 
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14. Clive, D., W.G. Flamm, and J.B. Patterson. 1973. Specific Locus 
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15. Crow, J.F. 1973. The Impact of Various Types of Genetic Damage. In 
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16. DeMars, R. 1974. Resistance of Cultured Human Fibroblasts and Other 
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17. DeSerres, F.J. 1975. Mutagens in Our Environment. Presented at the open 
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18. Drake, J.W. 1971. Mutagen Screening with Virulent Bacteriophages. In 
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97 


19. Fisher, H.W., and J. Yeh. 1967. Contact Inhibition in Colony Formation. 
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20. Fisher, R.A., H.G. Thornton, and W.A. MacKenzie. 1922. The Accuracy of 
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21. Flamm, W.G. 1975. Genetic Basis for Human Disease. Presented at the 
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22. Heidelberger, C. 1975. Chemical Carcinogenesis. In Annual Rev. Biochem. 
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23. Hitotsumachi, S., Z. Rabinowitz, and L. Sachs. 1971. Chromosomal 
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24. Hitotsumachi, S., Z. Rabinowitz, and L. Sachs. 1972. Chromosomal 
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25. Hsie, A.W., and T.T. Puck. 1971. A Morphological Transformation of 
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26. Juliano, R.L., and P. Stanley. 1975. Altered Cell Surface Glycoproteins in 
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27. Kao, F.T., and T.T. Puck. 1969. Genetics of Somatic Mammalian Cells. IX. 

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31. Legator, M.S. Personal Communication. 


98 


32. Malcolm, A.R. 1970. Toxicity of Divalent Cadmium to Mammalian Cells in 
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33. Malcolm, A.R., and R.R. Young. The CHO Cell/BrdU-Visible Light System 
as an Assay for Mutagens, (in preparation). 

34. Malcolm, A.R., B.H. Pringle, and H.W. Fisher. 1973. Chemical Toxicity 
Studies with Cultured Mammalian Cells. In Bioassay Techniques and 
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217-230. 

35. Malcolm, A.R., and K.O. Cooper. Unpublished Data. 

36. McCann, J., et al. 1975. Detection of Carcinogens as Mutagens: Bacterial 
Tester Strains with R Factor Plasmids. Proc. Natl. Acad. Sci. USA. 72: 
979-983. 

37. Nishioka, H. 1975. Mutagenic Activity of Metal Compounds in Bacteria. 
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38. Ofroth, G.L., and B.N. Ames. 1977. Mutagenicity of Inorganic Compounds 
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39. O’Neill, J.P., et al. 1977. A Quantitative Assay of Mutation Induction at 
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40. Patterson, D., F.T. Kao, and T.T. Puck. 1974. Genetics of Somatic 
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41. Pringle, B.H., et al. 1968. Trace Metal Accumulation by Estuarine 
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42. Puck, T.T. 1973. Genetic Biochemical Studies on the Mammalian Cell 
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44. Puck, T.T. 1972. The Mammalian Cell as a Microorganism. Holden-Day. 
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45. Shuster, C.N., and B.H. Pringle. 1969. Trace Metal Accumulation by the 
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46. Siminovitch, L. 1976. On the Nature of Heritable Variation in Cultured 
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47. Sirover, M.A., and L.A. Loeb. 1976. Infidelity of DNA Synthesis In Vitro : 
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100 


DEVELOPMENT OF A BIOASSAY FOR OILS 

USING BROWN ALGAE 


M. Dennis Hanisak 


Richard L. Steele 

U.S. Environmental Research Laboratory 
Narragansett, Rhode Island 02882 


ABSTRACT 

Bioassay procedures were developed to observe the effects of No. 2 fuel oil, 
two jet fuels, and a crude oil on the growth and early development of Fucus 
zygotes and Laminaria gametophytes. These algae are as sensitive, or more so, 
than fish and invertebrates previously tested in oil bioassays. Fucus sperm and 
Laminaria spores are extremely sensitive to oil, with dramatic effects at the 
levels of 2 ppb. These results indicated that these species are potentially good 
bioassay organisms, and also that chronic, low-level pollution could 
significantly alter the community structure in marine ecosystems. 

INTRODUCTION 

Algae are the primary producers in the marine ecosystem. Yet, despite their 
importance, little is known on how they are affected by specific pollutants in 
their environment. A bioassay is one method of studying pollution effects on 
organisms. This paper reports on bioassay procedures developed with two 
brown algal genera, Fucns and Laminaria. 

In order to be a useful bioassay organism, an alga should be readily available 
in nature, easily maintained in the laboratory, hardy enough to grow in culture, 
yet sensitive enough to respond to low levels of pollution encountered in 
nature, and preferably, of ecological or economic importance. Both Fucus and 
Laminaria have most, if not all, of these qualities; yet, because of differences in 
their habitats, they might react differently to an oil spill. Fucus, being an 
intertidal species, would be subjected to repeated immersion in the water mass 
and be coated with oil when the tide is out. Laminaria is a subtidal species and 
would normally be subjected only to concentrations of oil present in the water 
column. 


101 


Although Fucus and Laminaria are both brown algae, their life cycles are 
quite different. The life cycle of Fucus (Figure 8-1) is very much like that of an 
animal. Mature, diploid thalli produce haploid eggs and sperm which fuse to 
form zygotes that ultimately develop into new diploid thalli. The life cycle of 
Laminaria (Figure 8-2), in which an alternation of generations occurs, is much 
more complicated than that of Fucus. The microscopic, blade-like, diploid 
sporophyte alternates with a microscopic, Filamentous, haploid gametophyte. 
The variability and complexity of algal life cycles provide several opportunities 
to study the effect of a specific pollutant on growth and development. In the 
present study, the effects of different petroleum products on the growth of 
Fucus zygotes and Laminaria gametophytes were observed. 

MATERIALS AND METHODS 

Both Fucus and Laminaria plants were collected from various locations in 
and around Narragansett Bay (i.e., Camp Varnum, a National Guard 
installation, the dock of the Environmental Research Laboratory, Narragansett, 
R.I., and Monohan’s Cove, Narragansett, R.I.). In developing techniques, 
several species were used, including Fucus vesiculosus , F. edentatus , F. 
distichus. Laminaria saccharine, and L. digitalis. 

The Fucus species represent both monoecious and dioecious types. In 
deciding which species of Fucus and Laminaria to use, little difference was 
noted in preliminary response among various species. Data presented herein 
represent the responses of Fucus edentatus and Laminaria saccharina , but are 
representative of other species in both genera. 

For Fucus , methods of procurement of eggs and sperm were evaluated (4, 
5), and a technique was devised that is applicable to all species tested. The 
method is essentially a combination of other methods reported in the 
literature, and consists of the following: receptacles (fertile plant tips) that 
appear most erumpent and mature, even to the point of being partially eroded, 
were collected from mature plants. These receptacles were observed to produce 
the highest numbers of viable eggs and sperm. Receptacles were rinsed in sterile 
charcoal Filtered* seawater at 30 ppt. salinity, and were placed in a moist 
chamber overnight. The moist chamber consisted of large 150 x 25 mm plastic 
petri dishes (Falcon Plastics) containing filter paper of the same diameter 
moistened with sterile seawater. 


* Cartridge Filtration through Commercial Filter Corporation honeycomb 
wound Filters, 15 u porosity, and .22 u porosity pleated Gelman filters. All 
apparatus used in tests with both Fucus and Laminaria was plastic. 


102 


CONCEPTACLE SPERMATANGIA 



Figure 8-1. The Life Cycle of Fucus. 


SPOROPHYTE 


&GAMETOPHYTE 



SORI OF 
SPORANGIA 




SPERM 


9 GAMETOPHYTE 


SPOROPHYTE 

DEVELOPMENT 



FUSION 


Figure 8-2. The Life Cycle of Laminaria. 


103 















These plates were placed in a 12°C culture chamber overnight. Receptacles 
were then placed in sterile charcoal filtered seawater the next morning. Eggs 
and sperm were immediately released, and fertilization was observed within 15 
to 20 minutes. Zygotes were immediately pipetted into culture dishes (60 x 1 5 
mm plastic petri dishes, with 2 mm square grids — Falcon Plastics) while 
keeping the eggs suspended in seawater by stirring. 24 hours after dispensing 
into culture dishes, the toxicant was introduced by allowing the zygotes to 
settle on the bottom of the dish, the seawater removed, and replaced with 
seawater containing the toxicant at the test levels. In a few cases, the tips were 
pre-treated with the toxicant, and in these cases, the above sequence was 
suitably modified. 

The growth medium for the Fucus assays was, in all cases, sterile charcoal 
filtered seawater. The parameter measured for these experiments was the 
increase in length after 12 days of growth. 

Methods of procuring Laminaria spores were similar to those of Fucus 
gametes. Sporogenous plants were collected and then washed in deionized 
water to remove surface contaminants. Small pieces (2-3 cm square) of 
sporogenous tissue were placed into moist chambers overnight. These pieces 
were placed into sterile seawater the following morning, and spores were 
released in abundance. Spores were dispensed into culture dishes at 
concentrations sparse enough to allow counting and to prevent overcrowding, 
but dense enough (ca. 100-260 eggs) for good statistical data. In all cases, the 
culture medium was Provasoli’s Enriched Seawater (6). The parameter 
measured was the increase in diameter of the gametophyte after 21 days of 
growth. 

All assays were conducted at 400 ft-c of continuous cool white fluorescent 
light. Except for the first series of experiments to determine the optimal 
temperature salinity combinations for the assays, the temperature and salinity 
were 18°C and 30 o/oo for Fucus and either 12 or 18°C and 30 o/oo for 
Laminaria. For the various tests, the petroleum product (either No. 2 fuel oil, 
JP-4, JP-5, and Willamar crude) at concentrations ranging from 0-2000 ppm 
was equilibrated with seawater, proper dilutions made, and added to the 
cultures. The oil-seawater mixtures were analyzed by infrared 
spectrophotometry (Perkin-Elmer Model 621) to determine the amount of 
dissolved product causing toxicity. 

Observations and measurements of Fucus zygotes and Laminaria 
gametophytes were made with a Unitron inverted microscope (Model BMIC). 
Approximately 10-20 individuals were measured per dish. Four replicates were 
performed for each treatment. 


104 


RESULTS 


Fucus zygotes were much more tolerant of salinity-temperature extremes 
than were Laminaria gametophytes (Table 8-1). This is probably a reflection of 
their habitats; Fucus , being intertidal and growing in a more variable 
environment than the subtidal Laminaria , is adapted to a wider range of 
environmental conditions. Optimal growth of Fucus was at 18°C and 30-42 
o/oo while that of Laminaria was at 12 - 18°C and 24-36 o/oo. There appeared 
to be some seasonal variability in Laminaria's optimal temperature for growth. 
Fertile Laminaria collected during the colder winter months produced spores 
that germinated and grew slightly better at 12°C than at 18°C; the converse 
was true for Laminaria collected during the warmer spring months. In the 
following experiments to determine the toxicity of different oils, the standard 
conditions were 18°C and 30 o/oo for Fucus and 12°C and 30 o/oo for 
Laminaria. 

Of the four types of oil tested, No. 2 fuel oil was the most toxic to Fucus 
zygotes, and the jet fuels, JP-4 and JP-5, were least toxic (Table 8-2). There 
appeared to be a slight stimulation at lower levels (200 ppb or less), except for 
Willamar crude. This may be due to a surfactant effect on the part of the oil. 
Above those levels, these oils became increasingly deleterious to growth. 

The toxicity of the four oils to Laminaria gametophytes was similar to that 
of Fucus zygotes, although at lower levels, Laminaria response was not 
comparable to Fucus (Table 8-3). Number two fuel oil is still the most toxic, 
although not as relatively toxic as it was to Fucus. The jet fuels, JP-4 and JP-5, 
were somewhat more toxic to Laminaria than they were to Fucus. The lower 
growth rate of Laminaria gametophytes compared to Fucus zygotes is probably 
a reflection of their different growth habits. Laminaria gametophytes are much 
smaller, and grow in a more radial fashion than do Fucus zygotes. 

Preliminary experiments on application of oil during gamete release in 
Fucus , and spore release in Laminaria , indicate that these brown algae may be 
extremely sensitive to oils (Table 8-4). 

Concentrations greater than 20 ppb of No. 2 fuel oil were completely toxic 
to Laminaria spores. Even at 2 ppb, significant inhibition of the resulting 
gametophytes occurred. Fucus was even more sensitive. At 2 ppb, fertilization 
of eggs was blocked, apparently due to a toxicity effect on the sperm. 

DISCUSSION 

It is somewhat difficult to compare the results of these bioassays with 
brown algae, with those developed with other organisms by different 


105 



106 









Table 8-2. Effects of No. 2 Fuel Oil JP-4, JP-5 and Willamar Crude Oil 

on Growth of Fucus edentatus Zygotes. 

Numbers Shown are u in Length of Juvenile Plants 





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Dissolved hydrocarbons undetectable by spectrophotometry due to extremely minute amounts. Values are ranged of 
measurement and include all the petroleum products. 





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Table 8-4. Growth of Fucus Zygotes and Laminaria Gametophytes 
After Treatment in Oil/Water Mixture During Gamete and Spore Release. 

Treatment was with No. 2 Fuel Oil. 



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Spores germinated but were dead after day ni 




investigators using different techniques and response parameters. Based on the 
bioassays with Fucus and Laminaria , it appears that they were not as sensitive 
to oil as some microalgae that have been studied (7), but they were similar or 
slightly more sensitive than bioassays developed with fish and invertebrates (2, 
3). Although the toxicity values obtained in these studies were not directly 
applicable to Fucus and Laminaria (Pulich used doubling times and Eisler used 
LC 50 values) similar values can be derived. 

The extreme sensitivity of the reproductive stages, i.e., eggs and sperm in 
Fucus , and spores in Laminaria , indicate that either of these organisms might 
become a useful bioassay tool. 

In one experiment, Fucus receptacles were allowed to stand in various 
concentrations of oil 5 hours before being placed in moist chambers overnight. 
After completing the experiment in the usual manner, and allowing gamete 
release to occur in sterile seawater, the deleterious effects on the sperm were 
not observed. However, growth of the juvenile plants was reduced, being 
similar to that in Table 8-2, even though the zygotes were not in oil solutions 
during or after fertilizations. Further experiments with two-week old juvenile 
Fucus plants indicated that as the plant gets older, sensitivity to oils decreases. 
Thus, the most critical stage of the life cycle, and the one most sensitive to oil, 
is the reproductive phase. 

Continued development and refinement of these bioassay procedures is 
needed, as well as a survey of other seaweeds, to see how representative these 
results are for seaweeds in general. The development of a bioassay in a 
flow-through system would be more representative of natural conditions. 

The deleterious effects of low-level oil pollution on the reproductive cycle 
of these algae can easily be visualized, especially in areas of chronic pollution, 
such as those found near harbors, marinas, and similar installations. By 
preventing the completion of the life cycle, the community structure of algae, 
as well as that of higher trophic levels, will be altered. Greater effort should be 
made to examine chronic, long-term effects of oil pollution on the marine 
ecosystem. 


REFERENCES 

1. Dawson, E.Y. 1956. How to Know the Seaweeds. Wm. C. Brown, 
Publishers, Dubuque, Iowa. 

2. Eisler, R. 1975. Acute Toxicides of Crude Oils and Oil-Dispersant Mixtures 
to Red Sea Fishes and Invertebrates. Israel Journ. of Zool. 24:16. 


110 


3. Eisler, R., and G.W. Kissel. 1975. Toxicities of Crude Oils and 
Oil-Dispersant Mixtures to Juvenile Rabbit Fish, Signanas rivulatus. Trans. 
Am Fish. Soc. 104:571. 

4. McLachlan, J. 1974. Effects of Temperature and Light on Growth and 
Development of Embryos of Fucus edentatus and F. distichus ssp. 
distichus. Can. Journ. Bot. 52:943. 

5. Pollock, E.G. 1970. Fertilization in Fucus planta 92:95. 

6. Provasoli, L. 1968. Media and Prospects for the Cultivation of Marine 
Algae, p. 63 In A. Watanabe and A. Hatteri, Ed. Cultures and Collections of 
Algae. Proc. U.S.-Japan Conf. Hakone, Sept. 1966, Jap. Soc. Plant Physiol. 

7. Pulich, W.M., K. Winters, and C. Van Baalen. 1974. The Effects of No. 2 
Fuel Oil and Two Crude Oils on Growth and Photosynthesis of Microalgae. 
Marine Biology 26:87. 


Ill 



EFFECTS OF NO. 2 HEATING OIL 
ON FILTRATION RATE OF BLUE MUSSELS, 

MYTILUS EDULIS LINNE 


J. G. Gonzalez, D. Everich, J. Hyland, and B. D. Melzian 
U.S. Environmental Protection Agency 
Environmental Research Laboratory 
Narragansett, Rhode Island 02882 


ABSTRACT 

Reductions in gill filtration rates were observed for adult blue mussels, 
Mytilus edulis, that were exposed in a continuous flow-through dosing system, 
to three concentrations of the water-accommodated fraction of No. 2 fuel oil. 
The oil concentrations were measured routinely by infrared spectrometry, and 
averaged 0.019 ppm, 0.06 ppm, and 0.64 ppm throughout the exposure period. 
Filtering rates for healthy, unexposed mussels ranged from 7.2 to 30.9 ml/min, 
depending on ambient water conditions. In comparison to controls, filtering 
rates decreased as the oil concentration increased, with significant reductions 
occurring at all dose levels within 48 hours of exposure. Continued oil 
exposure up to two weeks produced progressively higher reductions in filtering 
rate. When returned to uncontaminated water for two weeks, the mussels 
resumed their normal feeding rates, revealing that the effect was reversible. 
Mussels collected from a small oil spill site exhibited similar responses. 

INTRODUCTION 

Bivalve mollusks are of considerable value to ecologists studying the effects 
of pollution; because many of the species are sedentary filter feeders, and are 
likely to accumulate contaminants from their surroundings. Mytilus edulis , the 
blue mussel, has become one of the most widely studied members of the group 
since it has a worldwide distribution; it is easy to maintain in the laboratory; 
and it is exploited commercially, particularly in European countries. Also, 
Mytilus , because of its intertidal existence, is particularly vulnerable to oil 
exposure. 

Several investigators have demonstrated a reduced feeding rate in mollusks 
exposed to environmental stress. Galtsoff et al (3) reported fifty percent 
reduction in gill ventilation rate of oysters exposed to an extract of crude oil. 


112 


They attributed this reduction to an anaesthetic effect upon the gill cilia. 
Preliminary investigations at the Environmental research Laboratory, 
Narragansett, revealed that feeding rates of clams, Mercenaria mereenaria , and 
scallops, Argopecten irradians, were notably diminished after exposure to No. 
2 fuel oil (7). 

A reduced feeding rate has also been noted for My tilus edulis. For instance, 
Abel (1) observed reduced filtration rates in mussels exposed to various 
pollutants including copper, zinc, mercury, cyanide, thiocyanate, and sulfide. 
Gonzalez and Yevich (5) reported that Mytilus show a significant decrease in 
filtering rate when exposed to high temperatures in the laboratory. Gilfillan 
(4), investigating the response of Mytilus to seawater extracts of crude oil, 
reported a decrease in both food consumption and assimilation, and an increase 
in respiration. The combined effects resulted in a reduction in the net carbon 
flux at oil concentration as low as 1 ppm. 

The Oil Pollution Research Branch at the Narragansett Environmental 
Research Laboratory has been assigned the task of evaluating the effects of 
very low levels of oil on ecologically and commercially important marine 
species. Such levels may not immediately lead to death of the organisms, but 
may ultimately jeapordize their long-term success at survival. Since Mytilus 
edulis is an important species in the marine community, and since a change in 
filtration rate appears to be a well-defined response to environmental 
disruption, we conducted an investigation to elucidate the behavioral effects of 
very low levels of oil, and to evaluate the recovery potential of the stressed 
animals. 

METHODS 

Adult Mytilus edulis were collected from the southeastern shore of 
Conanicut Island, Rhode Island, in April, 1976. In the laboratory, the animals 
were measured and separated into four groups of 50 individuals each. Mean 
shell length of the mussels was 4.63 cm. Each group of mussels was maintained 
for a two-week acclimation period in a plastic coated wire cage that was 
suspended in a one meter diameter fiberglass tank. The tanks were supplied 
with continuously renewed unfiltered seawater, which allowed the mussels to 
feed on natural plankton from the incoming water. 

After the acclimation period, the filtration rate was measured for each of the 
four groups of mussels. Next, these animals were placed for two weeks in a 
flow-through oil exposure system designed by Elyland etal. (6). One group was 
placed in each of three nominal oil concentrations-0.01 ppm, 0.1 ppm, and 1 
ppm- and one group was held under control conditions. Filtration rates were 
measured at various intervals during the two-week oil exposure period. All 


113 


animals were then transferred to control conditions, and their filtration rates 
measured periodically to determine recovery. 

Gill filtration rate was measured indirectly by recording the rate at which 
the animals removed food particles from the surrounding water. Each cage of 
50 mussels was transferred from its exposure or recovery tank to individual 
glass aquaria containing equal and known quantities of Isochrysis galbana. The 
algal suspension was maintained by aeration. Subsequently, three replicate 25 
ml water samples per aquarium were withdrawn at intervals during a three-hour 
feeding period. The number of algal cells in each sample was counted on a 
Coulter electronic cell counter, and the average percent reduction through time 
recorded for each aquarium. 

Filtration rates were expressed both graphically and numerically. Feeding 
curves were generated by plotting percent food particles removed versus time 
to allow visual comparison between the feeding rates of the control and oiled 
mussels for each of the various exposure or recovery periods. Results were 
analyzed statistically by performing linear regressions on the natural log 
transformed data, and comparing the regression lines (9). Actual filtration 
rate—the rate at which a solution is pumped through the gills of the animal in a 
given time period—was determined numerically with the aid of the following 
formula (8): 

Filtration rate (ml/min/mussel) = voL solution ( ml > x ln £o 

(no. animals) x A T min) C t 

where C Q and C t represent food concentrations at the beginning and end of a 
particular feeding interval (AT). The solution is based on the assumption that if 
filtration rate remains constant over the feeding interval, then the rate at which 
particles are removed from suspension will decline exponentially, as described 
by the curve e‘ x . For a given group of mussels, filtration rate was finally 
expressed as the average of those values calculated separately for each interval 
during which the mussels were actively removing food particles. Averaging is 
necessary to correct for the fact that the calculation of filtration rate can vary 
slightly depending on the magnitude of the time interval selected, a result of 
the fact that particles are not always removed at an exact exponential rate. 

The flow-through oil exposure system is designed to dose marine animals 
with the water-accommodated fraction of No. 2 fuel oil at three nominal 
concentrations-0.01 ppm, 0.1 ppm, and 1 ppm. The W.A.F. contains finely 
dispersed oil as well as the water-soluble components, but does not include the 
whole oil slick. The system simulates an area of chronic petroleum 
hydrocarbon pollution, such as one that might exist near a sewage outfall, an 
oil refinery, or an area consisting of sediments that have been heavily 
contaminated with oil. 


114 




Temperature, salinity, dissolved oxygen, and pH were routinely measured in 
the dosing tanks, and averaged 16°C, 31 ppt, 8.09 ppm, and 8.03 respectively. 
Hydrocarbon concentration was also determined routinely by infrared 
spectrometry, following the techniques described in Hyland, et al. (6). The 
actual oil concentrations measured according to this method, are somewhat 
different from the nominal ones mentioned previously. Accordingly, during the 
exposure period the 0.01 ppm tank averaged 0.019 ppm above the natural 
background hydrocarbon concentration; the 0.1 ppm tank averaged 0.06 ppm; 
and the 1 ppm tank averaged 0.64 ppm. 

RESULTS AND DISCUSSION 

Figure 9-1 illustrates the feeding activity of Mytilus edulis prior to oil 
exposure. There appears to be little difference in the shapes of the four feeding 
curves; and, in fact, statistical analysis revealed no significant differences (P < 
0.05). Typically, 80 percent of the food particles were removed by the mussels 
in 15 minutes, and 95 percent in 30-minutes, at which point maximum filtering 
activity was reached. Over this 30 minute interval, the average filtration rate 
for the four groups was calculated as approximately 18.1 ml/min, which is 
representative of values reported elsewhere in the literature (2). The values 
ranged from 15.6 for the control; to 18.7 for the 1 ppm group, and 19.1 for 
both the 0.01 and 0.1 ppm groups (Table 9-1). 



Figure 9-1. Pre-exposure: Comparison of Filtering Activity of 
Mytilus edulis Prior to Exposure to W.A.F. No. 2 Fuel Oil. 


115 









Table 9-1. Filtration Rates for Control and Oil-Exposed Mussels. 

NOTE: Mean and standard error (in parentheses) are both given. 



Control 

0.01 

ppm 

0.1 

ppm 

1.0 

ppm 

Pre-exposu re 

15.6 

19.1 

19.1 

18.7 


(0.4) 

(0.4) 

(1.9) 

(1.5) 

48-hr. Exposure 

19.3 

10.5 

5.2 

2.2 


(3.3) 

(0.9) 

(0.5) 

(0.2) 

2-wk. Exposure 

17.8 

5.3 

1.8 

0.3 


(1.5) 

(0.3) 

(0.5) 

(0.04) 

24~hr. Recovery 

15.8 

9.9 

3.9 

0.8 


(0.2) 

(1.2) 

(0.2) 

(0.1) 

2-wk. Recovery 

30.9 

— 

30.9 

17.2 


(6.5) 


(6.5) 

(1.6) 


After 48 hours of exposure (Figure 9-2), the filtration curves for the three 
experimental groups began to diverge, while the control curve retained 
pre-exposure characteristics. Mussel feeding activity in all three oil 
concentrations was significantly reduced from that of the control, with the 
highest concentration producing the most severe reduction. Filtration rates for 
the 0.01 ppm, 0.1 ppm, and 1 ppm polluted mussels, decreased to 10.5, 5.2 
and 2.2 ml/min, respectively, while the control group Filtered at an average rate 
of 19.3 ml/min. Figure 9-3 illustrates that continued oil exposure produces 
progressively lower Filtration rates. Mussels exposed for two weeks to 0.01 ppm 
required two hours to Filter what the controls Filtered in 30 minutes. Similarly, 
after three hours, animals exposed to 1 ppm had only consumed approximately 
35 percent of the algae; while the controls far surpassed this in less than 10 
minutes. After two weeks of exposure, filtration rates for the three exposed 
groups decreased to 5.3, 1.8, and 0.3 ml/min, while the control group Filtered 
at an average rate of 17.8 ml/min. 

Following the two-week exposure period, the animals were returned to clean 
water. Some evidence of recovery was noted after 24 hours in clean water 
(Figure 9-4); however, the filtration curves for all three exposure groups were 
still significantly different from the control. Filtration rates increased to 9.9 
ml/min for the 0.01 ppm group, 3.9 ml/min for the 0.1 ppm group, and 0.76 
ml/min for the 1.0 ppm group. 


116 






% Particles Removed from Water % p ar ticles Removed from Water 



Figure 9-2. 48-hour Exposure: Comparison of Filtering Activity 
of Mytilus edulis Exposed to W.A.F. No. 2 Fuel Oil. 



Figure 9-3. Two-week Exposure: Comparison of Filtering 
Activity of Mytilus edulis Exposed to W.A.F. No. 2 Fuel Oil. 


117 



















Figure 9-4. 24-Hour Recovery: Comparison of Filtering 
Activity of Mytilus edulis After 24 Hours of Recovery from 
Two Weeks Exposure to W.A.F. No. 2 Fuel Oil. 


Gradual improvement of all groups was observed as the animals remained in 
clean water. After two weeks, recovery was almost complete (Figure 9-5). The 
control and 0.1 ppm groups both Filtered at an accelerated rate of 30.9 ml/min, 
and the 1.0 ppm group Filtered at a rate of 17.2 ml/min, characteristic of 
pre-exposure rates. The higher Filtration rates observed may reflect increasing 
ambient water temperatures at the time of testing. Temperatures increased 
from 11°C at the time of pre-exposure testing, to 19°C during this latter 
testing period. Due to a laboratory failure resulting in reduced water flow, and 
subsequent anaerobic conditions in the recovery tank which held the 0.01 ppm 
exposure group, it was necessary to discard these mussels without 
demonstrating their complete recovery. However, since mussels at higher oil 
concentrations did recover, it seems reasonable to assume recovery for the 0.01 
ppm exposure group as well. There remained a signiFicant difference between 
the 1 ppm exposure group and the controls after two weeks in clean water; 
however, after one month of recovery, they actually fed slightly better than 
the controls. 

Based on the current investigation, it appears that the adverse effect of oil 
on Filtration rate of mussels is reversible, provided the stressed animals are 
returned to unpolluted conditions. However, the data also strongly suggest that 
recovery does not occur under conditions of continued exposure. Further 
investigation is currently in progress to determine the implications of reduced 


118 












Figure 9-5. Two-week Recovery: Comparison of Filtering 
Activity of Mytilus edulis after Two Weeks of Recovery from 
Two Weeks Exposure to W.A.F. No. 2 Fuel Oil. 

feeding over a long period of time as a result of continued oil exposure. A 
question confronted, for example, is whether mussels exposed for several 
months to chronic inputs of oil reveal reduced growth. 

The laboratory experiments reported herein were designed to investigate 
responses to chronic oil pollution, and not the acute phenomena which occur 
immediately after an oil spill. However, in November, 1976, a small spill of No. 
6 fuel oil occurred at Quonset Point, Rhode Island. The resulting slick drifted 
across the western passage of Narragansett Bay, and impacted approximately 
one mile of shoreline on Conanicut Island. This incident provided an 
opportunity to investigate the effects of spilled oil on filtering activity in 
mussels, and thus supports the laboratory results with field data. Accordingly, 
48 hours after the spill ,Mytilus were collected from the polluted site and from 
an unimpacted area nearby. Filtration rates were measured in the laboratory, 
and feeding curves were generated for both groups (Figure 9-6). Compared to 
controls, a small but statistically significant reduction in feeding activity was 
observed in oiled mussels. For example, over a period of 45 minutes, the 
controls had removed approximately 96 percent of the food particles, while 
the polluted mussels removed only 84 percent. Filtration rates were calculated 
as 7.2 ml/min (S.E. = 1.0) for the controls, and 4.9 ml/min (S.E. = 0.5) for the 
oiled group. The relatively low value obtained for the control group is most 
likely a reflection of low winter ambient water temperatures (5-6°C). One 


119 












Figure 9-6. Oil Spill: Comparison of Filtering Activity 
of Mytilus edulis Collected From a Clean Area and from an 
Area Impacted by an Accidental Spill of No. 6 Fuel Oil. 


week after the spill, another collection was made. Test results indicated that 
feeding activity of the oiled mussels had improved to the point that no 
differences could be found between control and oiled groups. 

In conclusion, the investigation demonstrates that (1) under laboratory 
conditions an adverse reduction in filtration rate occurs in Mytilus edulis at 
very low levels of continuous oil exposure; (2) the effect is reversible, since 
recovery will gradually occur if the stressed animals are returned to unpolluted 
conditions; and (3) a similar effect occurs in response to spilled oil in the 
natural environment. 

ACKNOWLEDGEMENTS 

Grateful appreciation is extended to Ms. Terry Richie and Dr. James Heltshe 
for their assistance with the statistical analyses. 

REFERENCES 

l.Abel, P.D. 1976. Effects of Some Pollutants on the Filtration Rate of 
Mytilus. Mar. Pollut. Bull. 7(12): 228-231. 


120 






2. Foster-Smith, R.L. 1975. The Effect of Concentration of Suspension on 
Filtration Rates and Pseudofecal Production for Mytilus edulis L., 
Cerastoderma edule (L.) and Venerupis pullastra (Montagu). J. Exp. Mar. 
Biol. Ecol. 17: 1-22. 

3. Galtsoff, P.S., H.F. Prytherch, R.O. Smith and V. Koehring. 1935. Effects of 
Crude Oil Pollution on Oysters. In: Louisiana Water, Bull. U.S. Bur. Fish. 
48(18): 144-210. 

4. Gilfillan, E.S. 1975. Decrease of Net Carbon Flux in Two Species of Mussels 
Caused by Extracts of Crude Oil. Mar. Biol. 29(1): 53-57. 

5. Gonzalez, J.G. and P. Yevich. 1976. Responses of an Estuarine Population 
of the Blue Mussel Mytilus edulis to Heated Water from a Steam Generating 
Plant. Mar. Biol. 34: 177-189. 

6. Hyland, J.L., P.F. Rogerson, and G.R. Gardner. 1977. A Continuous Flow 
Bioassay System for the Exposure of Marine Organisms to Oil, pp. 547-550. 
In. Proc. 1977 Oil Spill Conference (Prevention, Behavior, Control, 
Cleanup), March 8-10, 1977, New Orleans, LA. U.S. Coast Guard, 
Environmental Protection Agency, and American Petroleum Institute. 

7. Hyland, J.L., P.P. Yevich, and P.F. Rogerson. 1976. Unpublished Data. U.S. 
Environmental Protection Agency, Narragansett, Rhode Island. 

8. Quayle, D.B. 1948. Biology of Venerupis pullastra (Montagrie), Ph.D. 
Thesis, University of Glasgow. 

9. Snedecor and Cochran. 1967. Statistical Methods (6th ed.). Ohio State Univ. 
Press. Ames, Iowa. pp. 432-436. 


121 


LOBSTER BEHAVIOR AND CHEMORECEPTION: 

SUBLETHAL EFFECTS OF 
NO. 2 FUEL OIL 


Jelle Atema, Elisa B. Karnofsky 
and Susan Oleszko-Szuts 

Boston University Marine Program 
Marine Biological Laboratory 
Woods Hole, Massachusetts 02543 


ABSTRACT 

Lobsters (.Homarus americanus) were exposed in a flow-through oil dosing 
system to the water-accommodated fraction of #2 fuel oil. Behavioral 
observations of feeding efficiency and general behavior, showed that 5-day 
exposure to 0.08 and 0.15 ppm caused significant delays in feeding, without 
causing severe neuromuscular defects. Exposure to 1.5 ppm caused gross 
neuromuscular defects within 24 hours. Recovery was proportional to the 
gravity of observed defects. Neurophysiological experiments on antennular 
chemoreceptors of behaviorally observed animals showed that oil is perceived 
as a chemical stimulus, and can change normal responses to food juices. 
Oil-exposed animals show abnormal, bursting spike patterns, both 
spontaneously and in response to food juice. It remains to be proven that low 
level exposure effects are due to oil interference with chemoreception. 

INTRODUCTION 

Each year more evidence appears which demonstrates the importance of 
chemical signals in the lives of marine animals. The following are just a few 
examples of the broad categories of behavior where chemical signals are of vital 
importance: feeding behavior, both the predator’s detection of live prey and 
the scavenger’s localization of dead bait; the prey’s alarm and escape behavior; 
mating behavior and mate selection; parental brood recognition; and the 
selection of suitable geographic locations, as in larval settling and homestream 
return of migratory species. Interference with chemical signals or with the 
receptors that evolved to receive them could therefore jeopardize animal 
survival without causing immediately obvious deleterious effects on the 
individual. Man’s chemical discharges into the environment, such as large 
amounts of petroleum hydrocarbons in coastal areas, may cause such 
interference. 


122 


Speculations about petroleum hydrocarbon interference with the processes 
of chemoreception have appeared with a certain regularity in the literature, 
starting with Blumer (4). The reasons for this speculation are obvious: 
petroleum hydrocarbons are a mixture of organic chemical compounds, some 
of which are related to compounds such as pheromones and alarm substances, 
which are utilized by animals for their orientation and communication. These 
communication signals may have chemical features, such as carbon skeleton, 
functional groups, volatility, and solubility, in common with compounds in 
petroleum (7). In an oil-polluted environment, different petroleum compounds 
will be in solution or in emulsion in the water column, while the heavier 
fraction can become part of the benthic mud and affect the benthic ecosystem 
for many years, as shown by Blumer and Sanders, among others (5, 9, 10). One 
can thus envisage the scenario when these chemical look-alikes mimic or mask 
the reception of biologically important signals. Mimicked signals may result in 
“false alarm”, i.e., animals may look for imaginary food or mates, or avoid 
predator danger where there is none. If their chemical signals are masked, 
animals cannot respond to them and may miss opportunities to feed, or mate, 
or escape. A third possibility less frequently mentioned is that animals may 
become subject to two competing signals (3), for instance, an attractant signal 
from food (or mate) and a repellent signal from oil. In such cases 
chemoreception would be perfectly normal, but the animal may not be able to 
decide whether to feed or hide. Such delays may be more critical than apparent 
at first glance: even a slight delay in responding to food can put an individual 
at a significant disadvantage when competing with an unimpaired conspecific, 
or in escape from predators. 


Thus far, some cases of mimicked food attraction and delayed food 
responses have been observed, as well as increases and decreases in alarm and 
attraction behavior (1, 8, 11, 12). However, specific effects of oil on 
chemoreception itself have never been documented. Studies showing oil 
interference with chemoreception will provide us with a general understanding 
of the effects of oil pollution, since the processes of chemoreception — 
although essentially unknown — are probably similar in all animals. This would 
be especially true if similar effects for petroleum fractions were found in 
different animals. Interference with chemoreception or chemically mediated 
behavior also may be one of the most sensitive measures of low level oil 
pollution, since the much more obvious neuromuscular abnormalities appear at 
higher, although still sublethal, levels of oil exposure. 

For this study we chose the bait localization behavior of the lobster, 
Homarus americanus. The lobster uses two chemoreceptor organs. Aesthetasc 
hairs on the antennules represent their sense of smell, and function probably to 
detect distant chemical signals in low concentration. Hairs on the walking legs 
and maxillipeds are the equivalent of taste, and are essential in picking up food 
and bringing it into the mouth while testing its palatability for ingestion. In 


123 


this study, using the water-accommodated fraction (WAF) of #2 fuel oil, we 
are mainly concerned with distance chemoreception of the antennules. 

The First purpose of these experiments is to determine the range of #2 fuel 
oil exposures that affect the feeding behavior of lobsters without causing 
neuromuscular disturbance. Since chemoreception provides an important input 
into their feeding behavior, we then apply neurophysiological techniques to 
measure the effects of oil exposure on chemoreceptors in animals, where 
sublethal behavioral abnormalities have been shown. This is the second goal of 
these experiments. 

MATERIALS AND METHODS 

Flow-Through Oil Dosing System 

In order to measure actual exposure levels, a continuous flow-through oil 
dosing system is necessary. The flow-through system (Figure 10-1) consisted of 
two head tanks, one control and one experimental. The experimental head 
tank, 8’ x 11” x 8”, was fitted with three baffles to aid in the layering of the 
oil after mixing. Its inflow was 4,000 ml/min. Oil was introduced via a syringe 
pump at a fixed rate into the center of the fast jet of seawater, causing rapid 
emulsification. The overflow of the head tank was skimmed off into a 
collecting tank where the oil layer was siphoned off occasionally. From the 
head tank, the oil-water mixture entered six 100-liter tanks individually. 

The overflow from the individual tanks entered a holding tank where 
lobsters were stored for neurophysiological preparations on oil-exposed 
animals. The overflow from the collecting box and the holding tank entered an 
acrylic-fiber filter box, where oil was removed before the water entered the 
drain (Figure 10-1). The control head tank, 4’ x 11” x 8”, supplied four 
individual 100-liter tanks. Its inflow was 2,600 ml/min. Individual tanks, both 
experimental and control, had inflows ranging from 400-460 ml/min. Water 
quality — salinity, temperature, ammonia, pH and 0-> content — and flow rate 
to individual tanks were measured every other day. 

Behavior 

Two male and two female lobsters served as controls, three males and three 
females as experimental. From our holding facility, we chose lobsters which 
had molted within two to eight weeks of the start of the experiment, to avoid 
effects of pre-molt behavior during observation. The animals were measured for 
close size match, and put in individual tanks containing a glazed clay shelter 
and a pebble substrate. They were fed twice daily until all animals were feeding 
normally. Then a base line for feeding behavior was determined over a five-day 
period. During the whole experiment lobsters were observed daily in the early 
morning (7-9 am) and late afternoon (4-6 pm). One-minute behavior recordings 
were followed by the addition of food, which was lowered on a string from the 
right or left front corners, alternately. Apart from general behavior (about 25 


124 


INFLOW 



Figure 10-1. Diagram of Flow-through Oil Dosing System. 

NOTE: See text for details. 


different postures and movements) the times for alert (first observable response 
to food), wait (period between alert and leaving shelter to search), and search 
(time after first leaving shelter until food touched with maxillipeds) were 
recorded. On the morning of the 6 th day, #2 fuel oil was introduced at a 
predetermined flow rate. Lobster behavior was recorded 6 hours later and 
subsequently twice daily, as above. After five days, oil introduction was 
stopped. All recordings were continued as before for another five days to 
determine behavioral and chemical recovery rates. 

Oil Chemistry 

Concentrations of total hydrocarbons in the water column were determined 
by infrared spectroscopy before, during, and after introduction of oil. On the 
day before oil was added, a 2-1 water sample from every tank was extracted 
with 50 ml CClq. A second extraction was performed 12 hours and a third 24 
hours after onset of oil mixing. Extractions were continued daily for the 
remaining four days of oil exposure, and on the first and second day 
post-exposure to determine how quickly oil left our system. A small number of 
CH 0 CI 9 extractions were performed for gas chromatographic analysis. The oil 
used in the study was an Exxon #2, provided by the EPA Environmental 
Research Laboratory, Narragansett, Rhode Island. 


125 





































































































Figure 10-2. Diagram of Stimulation-Recording 
Chamber (“Olfactometer"). 


NOTE: Stimulus is injected with a syringe (A) into a seawater flow (B) over 
the dissected lobster antennule. The antennule is perfused through a micro¬ 
pipette with oxygenated lobster saline (C) which exits the antennule into a 
bath of saline (D). To make recordings, one small bundle of nerve fibers is 
lifted from the saline bath into the air with a platinum hook electrode (E). 
Seawater and saline baths are separated by a Sylgard cork, through which the 
antennule passes. 


Neurophysiology 

Electrophysiological data were obtained from chemoreceptors of oil-exposed 
and normal lobsters, some of which had been observed behaviorally. This 
permits a comparison between the neural chemosensory input the animal 
received, and the resultant behavior after processing through higher nerve 
centers. Such a comparison is a necessary step in determining whether oil 
interferes with behavior through chemoreception. 

To measure neurophysiological activity, the lateral flagellum of the 
antennule of a lobster was removed and placed in fresh seawater. The cut 
proximal end was inserted through a Sylgard cork. Three to four cuticular rings 
were removed. The distal tip was cut and the antennule placed in a lobster 
saline bath in the stimulation-recording chamber (Figure 10-2). A micropipette 
was inserted snugly into the distal tip, and perfusion with oxygenated lobster 





























saline started within 10 minutes after the removal of the antennule from the 
animal. Test chemical stimuli were injected into the continuously flowing 
saline bathing the antennule. Recordings were made by picking up a small 
nerve bundle with a platinum electrode. The signal was amplified via a 
Tektronix Type 122 Preamplifier, and displayed on conventional recording 
equipment for later analysis. 

It is commonly accepted that neurophysiologically determined thresholds of 
sensory receptors lie an order of magnitude above the behaviorally determined 
thresholds. Thus, to document the effects of #2 fuel oil on lobster antennular 
chemoreception, we used the following test series: (1) mussel juice; (2) #2 fuel 
oil, 10 ppm: (3) mussel juice plus oil; (4) artificial seawater; and (5) mussel 
juice. Stock solutions were made at one time and refrigerated. Artificial 
seawater was made according to the MBL formula: 420 mM NaCl, 9 mM KC1, 
9 mM CaCl 2 *2H 2 0, 23 mM MgCly6H 2 0, 26 mM MgS0 4 '7Ho0, 2 mM 
NaHCO^ (pH 7.3). This was used to eliminate introduction of day-to-day 
variations in natural seawater. Mussel juice was made by homogenizing 10 g 
wet weight o {Mytilus edulis tissue in 100 ml artificial seawater. The suspension 
was centrifuged at 27,000xg for 20 minutes and the pellet discarded. The 
supernate was frozen in small aliquots until needed. The WAF of 10 ppm #2 
fuel oil was made at the start of each preparation, due to the lability of the 
oil-water suspension. 

This protocol allowed us to compare the response to Stimulus (1) with the 
response to Stimulus (5) for nerve fiber damage or fatigue, or lasting effects of 
the prior oil test stimulus. Stimulus (2) and Stimulus (3) were used to 
determine a) whether lobster antennules can detect oil as a chemical stimulus, 
and b) if the presence of oil changes the response to mussel juice. Stimulus (4) 
was used to determine the sensitivity of the preparation to a chemically neutral 
stimulus; this allowed us to measure mechanoreceptor activity which can be 
subtracted to discover purely chemosensory responses in the other tests. 


RESULTS 

Chemistry 

Water quality measurements showed that for all experiments, salinity and 
pH remained constant, ammonia remained undetectably low, and remained 
at saturation. Temperatures fluctuated with ambient water temperature, and 
are listed below with each experiment. 

Gas chromatography showed that the water-accommodated fraction 
recovered from the lobster tanks closely resembled whole #2 fuel oil. Infrared 
spectroscopy of CCLq-extractable lipids showed moderate (±20%) daily 
fluctuations within and between individual tanks. The dosing system was 
capable of maintaining relatively similar exposure levels. 


127 


Behavior 


In the first experiment, the recovered oil level in the exposure tanks waj 
about 0.08 ppm total hydrocarbon. The temperature gradually rose from 22° 
to 24.5° C over the 15-day experimental period. Behavioral changes were 
observed in the morning alert times, which, when comparing oil-exposed days 
with pre-exposure days, slowed in experimental animals (p < 0.05). Total food 
localization time was also slower (p < 0.025), perhaps as a result of slower 
alerting. Control animal behavior did not change (Table 10-1). In this first 
experiment, defensive postures and sometimes erratic and frantic behavior was 
observed in most of the exposed lobsters, and not in control animals. Defensive 
postures are characterized by wide open seizer claws, held close to the body, 
while the animal sits retreated far into its shelter. Erratic and frantic 
movements are sudden, unprovoked seizer snapping, jerky body movements 
and twitches. 

In the second experiment, the recovered oil level was about 0.15 ppm; 
temperature was a constant 10° C. Neither oil-exposed nor control animals 
showed significant changes in feeding behavior in the morning obervation, 
when comparing pre-oil with oil exposure period. In the afternoon observation, 
oil-exposed animals did not change their search speed but their alert was 
delayed (p < 0.05). Control animals had a faster search time in the afternoon 
(p < 0.005). Both control (p < 0.01) and experimental animals (p < 0.05) 
showed shorter wait times (Table 10-1). 

In the third experiment the temperature rose from 11° to 13.5° C, and the 
recovered oil level was 1.5 ppm. At 30 hours the lobsters showed gross 
neuromuscular defects, and oil inflow was stopped. In this experiment, 
behavior in post-exposure recovery period was compared with pre-exposure 
behavior. Experimental lobsters were slower in all phases of feeding behavior 
during the five-day recovery period than in the five-day pre-oil period 
(p < 0 .001). Even five days after exposure to 1.5 ppm #2 fuel oil for 30 hours, 
half the lobsters did not feed within the 10-minute limit (Figure 10-3). Control 
lobsters showed no significant differences (Table 10-1). The animals during this 
last experiment showed three levels of effects, some animals being affected 
much more than others. A description of the tliree levels follows (see also 
Figure 10-3). 

• Most extreme (two lobsters) —After 30 hours of oil exposure, these 
lobsters were found outside their burrows lying on their backs, pleopods 
twitching or still, tail half curled, walking legs twitching, antennae and 
antennules limp, gill bailers moving slightly. Occasionally the back and tail 
were arched and then curled. At times, attempts were made to right itself. 
Body jerked after food entered into tank. No recovery occurred in five 
days. 


128 


Table 10-1. Significant Changes in Feeding Behavior of Lobsters 
Before and During Exposure to #2 Fuel Oil (WAF)*. 


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129 


the pre-exposure control period. Significance levels are given in 
parentheses. 


RECOVERED OIL (PPM) 


EFFECT OF NO. 2 FUEL OIL ON 

LOBSTER FEEDING BEHAVIOR z o 



PRE OIL POST 


Figure 10-3. Effect of 1.5 ppm No. 2 Fuel Oil (WAF) 
on Lobster Feeding Behavior. 

NOTE: Large bars indicate number of lobsters not feeding within 10 minutes 
of food introduction. Broken line represents estimated oil levels in lobster 
tanks based on actual recovery measurements (indicated by dots and stand¬ 
ard deviation bars). Hatched horizontal bar shows period of oil introduction 
into the system. "Pre" is the 5 day pre-exposure period: all lobsters feed and 
recovered oil remains below 0.05 ppm. "Oil" is the exposure period: highest 
exposure level 1.7 ppm, oil stopped after 30 hrs. "Post" is the recovery per¬ 
iod: oil quickly leaves the system, feeding behavior remains seriously affected 
for the entire 5-day period. 


• Moderate (three lobsters) — These animals were generally out of their 
burrows in two alternating stances. In one, the tail and head were down, 
the antennae were folded back, the antennules beat slowly, and the 
animal lay down low on the walking legs. This appeared to be a resting 
position. Then the tail and head arched up, the claws were opened and 
held close to the body, the antennae and antennules were held straight up 
and together, animal stood high on walking legs, and made frequent tail 
flips. Poor coordination was exhibited. Frequent aggressive lunges were 
made with open, raised seizer claw, or jabs with both claws at no obvious 
target. Lobsters were unaware of the presence of food, at times walking 
right over it without responding. If they did pick up the food, they 
continued to wander aimlessly around the tank with the food clutched 
tightly in the maxillipeds. Recovery in five days was almost complete in 
two animals; one animal did not recover. 

• Light (one lobster) — This animal remained in its burrow but was high on 
its walking legs, “spider” position and shaking. There were sporadic alerts 
not necessarily related to food. It exhibited no search. After two days 
recovery, there were slow hesitant approaches to the food with tail flips at 
the slightest irregularity. At the end of five days there was complete 
recovery. 


130 















































Neurophysiology 

The preliminary results from neurophysiological experiments on the 
lobster’s olfactory chemoreceptors are presented with a few examples in Figure 
10-1. Details are provided in the figure legend. These examples show that (1) 
the water-accommodated fraction of #2 fuel oil itself can be perceived as a 
stimulus by the primary receptor cells, (2) that the presence of oil in a mussel 
juice food stimulus can change the response pattern of the small nerve bundle, 
and (3) that exposure to oil causes abnormal bursting patterns (See Figure 
10-4). 

Generally, differences in chemoreceptor responses between mussel and 
mussel-plus-oil are more distinct in oil-exposed lobsters than in controls. One 
other striking feature is the tendency of oil-exposed individuals to exhibit 
irregular bursts, or frequent small clusters, of spikes, both spontaneously and in 
response to stimuli (Figure 10-4), This may be a general injury response (6) 
here caused by oil exposure. However, it has also appeared in nerves from 
animals which were exposed to very low levels of oil (0.3 ppm) in response to 
mussel-plus-oil stimuli, but not to mussel alone. 

DISCUSSION 

Our experiments have shown thus far that the original hypothesis that oil 
pollution may interfere in a number of different ways with chemoreception, 
and hence marine animal behavior, is not unreasonable. Behavioral experiments 
on the efficiency of the lobster’s chemically mediated feeding behavior have 
shown that exposure to #2 fuel oil (WAF) causes significant delays after five 
days at exposure levels as low as 0.08 and 0.15 ppm. Increased dosage caused 
increasingly severe effects. Also behavioral recovery was a function of exposure 
level. At higher exposures (1.5 ppm) serious neuromuscular abnormalities 
appeared within 30 hours. Lobsters showed great individual differences in 
behavioral effects and recovery. The range of exposure levels where behavior 
was affected, but no serious neuromuscular defects appeared, proved to be 
surprisingly narrow. However, further experiments are required for complete 
documentation of this point. 

Parallel neurophysiological experiments on the effects of such exposures on 
chemoreceptor performance showed that the receptors perceive oil as a 
chemical stimulus, that the presence of oil could modify normal responses, and 
that oil-exposed lobsters ofen showed abnormal receptor activity, both 
spontaneously and in response to food stimuli. 

Based on these results, it appears that #2 fuel oil (WAF) interferes with 
lobster behavior in a number of ways. At low exposure levels (0.1 ppm range), 


131 


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of Antennular Chemoreceptors to Chemical Stimuli. 


132 




















the observed effects on behavior may be due to oil-induced changes in 
chemoreception. At higher exposure levels (1 ppm range), the behavioral 
effects appear neuromuscular, with a loss of coordination and equilibrium. 

These preliminary results are being corroborated at different exposure levels. 
In additional experiments, we will attempt to provide a correlation between 
behavioral and neurophysiological results of oil exposure to determine if the 
observed behavioral effects of oil are caused by a malfunctioning 
chemoreceptor system. 

ACKNOWLEDGEMENT 

We would like to thank L. Ashkenas, B. Bryant and T. Dourdeville for 
expert technical assistance. We are indebted to B. Melzian and D. Stenzler for 
help in designing and construction of the flow-through oil dosing system. Dr. 
B. Ache and B. Johnson provided us with the design and training for valuable 
discussions during the course of these experiments. Financial support was 
provided by grants from the U.S. Environmental Protection Agency 
(R-803833) and the U.S. Energy Research and Development Administration 
(E(l 1-1)2546). 

REFERENCES 

1. Atema, J. 1976. Sublethal Effects of Petroleum Fractions on the Behavior 
of the Lobster, Homanis americanus, and the Mud Snail, Nassarius 
obsoletus. In: Estuarine Processes; Uses, Stresses and Adaptation to the 
Estuary, Wiley, M. (ed.), Academic Press, New York, Vol. 1. pp. 302-312. 

2. Atema, J. 1977. The Effects of Oil on Lobsters. Ocean us 20: 67. 

3. Atema, J. and L. Stein. 1974. Effects of Crude Oil on the Feeding Behavior 
of the Lobster, Homanis americanus. Envir. Poll. 6: 77. 

4. Blumer, M. 1970. Oil Contamination and the Living Resources of the Sea. 
FAO Technical Conference on Marine Pollution and Its Effects on Living 
Resources and Fishing, Rome, December 9-18. 

5. Blumer, M. and J. Sass. 1972. Oil Pollution: Persistence and Degradation of 
Spilled Fuel Oil. Science 176: 1120. 

6. Dethier, V.G. 1971. A Surfeit of Stimuli: A Paucity of Receptors. Amer. 
Sci. 59: 706. 


133 


7. Jaenicke, L., D.G. Muller and R.E. Moore. 1974. Multifidene and 
Aucantene, C-ll Hydrocarbons in the Male-Attracting Essential Oil from 
the Gynogametes of Cutleria multifida (Phaeophyta). J. Amer. Chem. Soc. 
96: 3324. 

8. Mitchell, R., S. Fogel and I. Chen. 1972. Bacterial Chemoreception: An 
Important Ecological Phenomenon Inhibited by Hydrocarbons. Water Res. 
6: 1137. 

9. Sanders, H.L., J.F. Grassle and G.R. Hampson. 1972. The West Falmouth 
Oil Spill. I. Biology. Woods Hole Ocenographic Institution 72-20. Unpubl. 
Manuscr. 

10. Sanders, H.L., J.F. Grassle, G.R. Hampson, L. Morse and S. Garner-Price. 
1977. Anatomy of an Oil Spill: West Falmouth Study. Submitted to 
United States Environmental Protection Agency. 574 pp. 

11. Takahashi, F.T. and J.S. Kittredge. 1973. Sublethal Effects of the Water 
Soluble Component of Oil: Chemical Communication in the Marine 
Environment. In: The Microbial Degradation of Oil Pollutants, Ahearn, 
D.G. and Meyers, S.P. (eds.). Publ. No. LSU-SG-73-01. Center Wetland 
Resources, Louisiana State Univ., Baton Rouge, La. 259 pp. 

12. Walsh, F. and R. Mitchell. 1973. Inhibition of Bacterial Chemoreception by 
Hydrocarbons. In: The Microbial Degradation of Oil Pollutants, Ahearn, 
D.G. and Meyers, S.P. (eds.). Publ. No. LSU-SG-73-01. Center Wetland 
Resources, Louisiana State Univ., Baton Rouge, La. 275 pp. 


134 


INFLUENCE OF NO. 2 FUEL OIL 
ON SURVIVAL 
AND REPRODUCTION 
OF FOUR MARINE INVERTEBRATES 

J. A. Pechenik, D. M. Johns, and D. C. Miller 
Environmental Research Laboratory 
U.S. Environmental Protection Agency 
Narragansett, Rhode Island 02882 


ABSTRACT 

Responses to the water accommodated fraction of No. 2 fuel oil were 
determined in three marine gastropods (Nassarius obsoletus, Crepidula 
fornicata and Urosalpinx cinerea), and one Crustacean ( Cancer irroratus'). 
Experiments were conducted in either flowing or static systems at the 
following nominal oil concentrations: 0.0 ppm (control), 0.01 ppm, 0.1 ppm, 
1.0 ppm. Mortality of adults and larvae was consistently pronounced only at a 
nominal concentration of 1.0 ppm. Toxicity to adult TV. obsoletus at this 
concentration was greater during the winter than during the summer. Presence 
of sediment accelerated mortality during the summer, but had no effect on 
winter mortality. Exposure of adult N. obsoletus and U. cinerea to oil 
concentrations as low as 0.01 ppm and 0.1 ppm, respectively, interferred with 
normal patterns of egg capsule deposition. Exposure to oil did not alter the 
number of eggs/capsule in N. obsoletus or U. cinerea, and embryos produced 
by oil-exposed snails were viable. Fecundity of N. obsoletus may be reduced at 
a nominal concentration of 0.10 ppm. Growth rates of larval N. obsoletus and 
C. fornicata were reduced at nominal levels of 0.01 ppm and greater. Larvae of 
C. irroratus reared at a nominal concentration of 0.1 ppm weighed less at all 
zoeal stages relative to controls, even though carapace length of each larval 
stage, and time required to reach the megalops stage of development, were not 
altered. 

INTRODUCTION 

Lethal effects of petroleum hydrocarbons have been documented for a 
« variety of marine organisms (21), including zooplankton (20) and both adult 
and developmental stages of benthic invertebrates (2, 9, 11, 19). Sublethal 
concentrations of hydrocarbons are also known to interfere with aspects of 


135 


invertebrate reproduction, such as sperm motility and fertilization success (22, 
25), and embryonic cleavage rates (1, 22). Reduced egg production has been 
reported for oil-exposed Mytilus edulis (7) and Eurytemora affinis (5), and 
development of some larval Crustacea (16, 32, 33) and bivalves (9, 24, 25) is 
delayed after exposure to sublethal oil concentrations. Oil-induced changes in 
larval behavior of Homams americanus (32) and Cancer irroratus (6) have also 
been demonstrated. Recently, Anderson et al (3) have reported effects of low 
hydrocarbon levels on hatchability of fish embryos, and on heart beat rate of 
larval fish. 

The general objective of this investigation was to elucidate some sublethal 
effects of No. 2 fuel oil (introduced as the water accommodated fraction, 
WAF) on aspects of the reproductive and developmental biology of several 
common coastal invertebrates. Specific topics studied include egg capsule 
deposition, fecundity, hatchability and larval growth rates. The lethal dose of 
the oil was also determined for adult and larval Nassarius obsoletns, and larvae 
of Crepidula fornicata and Cancer irroratus , in order to establish sublethal 
exposure levels. 

A review by Moore and Dwyer (21) suggest that larval organisms are more 
sensitive to hydrocarbon toxicity than are adults, yet tolerance data on adults 
and larvae of the same species are infrequently reported. Culliney et al (12) 
suggest that the high surface/volume ratio in larvae and their “obligatory 
exposure to whatever may be in the water” would make larvae particularly 
susceptible to toxic substances, such as oil, at very low concentrations. Our 
study includes work on both adults and larvae of N. obsoletns, to further 
examine this hypothesis. 


MATERIALS AND METHODS 

Experiments were conducted using the gastropods Nassarius obsoletus, 
Crepidula fornicata and Urosalpinx cinerea , and the crustacean Cancer 
irroratus. Adults were exposed to oil using the flow-through oil-dosing system 
described by Hyland et al (15). Briefly, unfiltered seawater and No. 2 fuel oil 
enter a mixing chamber. The WAF produced is then metered into exposure 
tanks where it is diluted to the desired concentration by controlled flow of 
untreated seawater. Total hydrocarbon concentrations in control (for 
background) and experimental tanks are monitored three times/week by 
infrared spectrophotometry, and flow rates are adjusted to maintain desired 
WAF exposure levels. Nominal total hydrocarbon concentrations (WAF) were: 
0.0 ppm (control), 0.01 ppm, 0.1 ppm, and 1.0 ppm. Because measured 
hydrocarbon concentrations varied with time in the flow-through system, the 
nominal concentrations (cited as “X” ppm in the text) indicate only the order 


136 


of magnitude dose level employed. Mean total hydrocarbon concentrations (± 
s.d.) measured during each experiment are given with the results; between 
treatment, mean hydrocarbon values were significantly different for all 
experiments (P < 0.05). 

All three gastropod species studied produce egg capsules. While C. fornicata 
broods its capsules, U. cinerea and N. obsoletus attach capsules to solid 
substrates and then abandon them, making these capsules easy to collect and 
count. Descriptions of larval development have been published for N. obsoletus 
(29), C. fornicata (34) and C. irroratus (28). 


Adult Mud-Snail Survivarship and Egg Capsule Deposition 

Experiments with adult mud-snails were conducted in the flow-through 
dosing system described above. Adults of N. obsoletus were collected from 
Bissell Cove, Rhode Island, and groups of 35-100 individuals placed in circular 
plastic containers (26 cm diameter, 6 cm high) and completely submerged in 
the dosing tanks. The top and sides were perforated to permit water 
circulation. Surface area of the top and side of each container was 
approximately equal. Snails were fed shredded Mercenaria mercenaria tissue 
weekly, and the number and position of deposited egg capsules were recorded 
before capsules were removed each week. All container surfaces were wiped 
clean after each examination. Dead snails were counted and removed 
periodically. The mean number of eggs per capsule was determined for N. 
obsoletus in all treatments. Since N. obosoletus is primarily a deposit feeder 
(30), and sediments are known to accumulate petroleum hydrocarbons from 
seawater (15, 17), one experiment was run with mud added to evaluate its 
influence on toxicity. 

Reproduction of Urosalpinx cinerea 

Specimens of U. cinera were collected at Jamestown, R.I., in May, 1976, 
and groups of ten individuals were placed in perforated plastic freezer 
containers. Three boxes were submerged in the flow-through system at each of 
the following nominal oil levels: control (0.0 ppm), 0.01 ppm, 0.1 ppm. 
Freshly collected barnacles were provided weekly as food. Once each week, 
deposited egg capsules were counted and then removed. In July, a sample of 
egg capsules was taken from each treatment level to determine the mean 
number of eggs encapsulated. The number of females present in each container 
was determined in the middle of the experiment using the live-sexing technique 
of Hargis (13). 


137 


Larval Survival and Growth 


Survival of larvae exposed to No. 2 fuel oil (WAF) was assessed primarily 
under static conditions. Glass scintillation vials were completely filled with 
water siphoned from the flow-through dosing tanks. 15 to 20 two to three day 
old N. obsoletus or C. fornicata larvae were pipetted into each vial, and 
Isochrysis galbana was provided as food at an initial density of about 1x10 
cells/ml. Vials were then tightly capped to minimize volatilization of 
hydrocarbons. Each experiment was conducted in triplicate. Larvae were 
counted daily and survivors were transferred to fresh medium. The experiments 
were conducted at room temperature (21-23°C), well within the range for 
good larval growth (10, 31). 

One preliminary flow-through experiment was conducted with about 100 
two-day old C. fornicata larvae (approximately 420 Aim in shell length), using a 
system similar to that described by Calabrese and Rhodes (10). Water from the 
oil-dosing tanks was siphoned at approximately 50 ml/minute into 
flow-through chambers containing larvae, and 200 ml /. galbana suspension 
added at the beginning and end of each day as a feeding supplement. 

Completely filled, capped quart glass jars were used to determine the effects 
of the oil on growth and survival of larval C. irroratus. 75 Stage I zoea were 
added to each jar. Larvae were fed Artemia salina nauplii provided in excess 
numbers. Larval mortality was determined daily, and survivors were transferred 
to fresh medium. These experiments were conducted at 15°C, the optimal 
temperature for development of C. irroratus (28), and under a 12L:12D 
photoperiod. In a separate series of experiments, at least five individuals of 
each larval stage were harvested from control and “0.1” ppm levels to monitor 
growth. Larval carapace lengths were measured using an ocular micrometer. 
Dry weights were then determined using a Perkin-Elmer Electrobalance after 
the larvae were rinsed with distilled water, and dried at 80°C for 24 hours in 
pre-weighed foil pans. 

RESULTS 

Adult and Larval Survival 

Exposure to a nominal concentration of 1.0 ppm was found to be lethal for 
all adults and larvae tested. Concentrations of “0.1” ppm and “0.01” ppm 
were sublethal to all test organisms for the particular exposure periods of our 
experiments. 

Mortality of N. obsoletus adults did not exceed five percent in any 
experiment at control, “0.01” ppm or “0.1” ppm exposure levels. Substantial 


138 


mortality occurred only at “1.0” ppm. Pronounced seasonal variation in 
toxicity was evident (Figure 11-1). In one winter exposure, mortality at “1.0” 
ppm reached 50 percent within approximately 30 days, with the remaining 
snails dying during the subsequent 30 days. Similar results had been obtained 
in a preliminary experiment initiated the preceding February. A very different 
mortality profile was seen at “1.0” ppm in two summer exposures. In 1976, 
approximately 40 percent of the snails were still living at the end of three 
months (Figure 11-1). In 1977, presence or absence of sediment in the holding 
containers was added as another variable. While summer toxicity of the WAF 
was still relatively low, mortalities were substantially increased by the presence 
of mud (Figure 11-2). In contrast, the mortality pattern was unaffected by 
sediment in the winter. 



Figure 11-1. Survival of adult N. obsoletus exposed to 
"1.0" ppm No. 2 fuel oil (WAF). 


NOTE: Control mortalities were less than five percent. Winter experiment was 
run 10/22/76 - 12/15/76 (100 snails/treatment). Summer experiment was run 
7/6/76 - 10/12/76 (20 snails/treatment). Mean oil hydrocarbon concentrations 
± s.d. (N measurements) were: 1.25 ppm ±0.34 (25), winter; 0.94 ppm ±0.44 
(31) summer. 


139 






100 


• NO MUD PROVIDED 
O MUD PROVIDED 



DAY 


Figure 11-2. Survival of adult N. obsoletus exposed to 
"1.0" ppm in the presence or absence of sediment. 


NOTE: Winter exposures were initiated 10/22/76 (50 snails/treatment). Sum¬ 
mer exposures were initiated 7/12/77 (25 snails/treatment). Control mortal¬ 
ities less than 5 percent. Mean oil hydrocarbon concentrations ± s.d. (N) were: 
1.25 ppm ±0.34 (25), winter; 0.70 ppm ±0.27 (23), summer. 


Larval mortality of N. obsoletus was high in four to eight day experiments 
at “1.0” ppm, and relatively low at lesser concentrations (Table 11-1). 
Substantial batch variability in larval tolerance was observed (Figure 11-3). 
Whereas 50 percent mortality was recorded at “1.0” ppm after three-days in 
experiment “A”, the 50 percent level was not exceeded until day eight in a 
second experiment using a different hatch of larvae. Indeed, no mortality 
occurred until day four in experiment “B”. 

All C. irroratus larvae exposed to “1.0” ppm died within four days. At 
“0.1” ppm and “0.01” ppm, about 33 percent of the larvae were still living 
after three weeks (Figure 11-4). Survival to the megalops stage, attained after 
25-28 days, was: control, 58%; “0.01” ppm, 34%; “0.1” ppm, 30%; “1.0” 
ppm, 0%. 


140 





Table 11-1. Larval Mortality of N. obsoletus after Exposure to No. 2 Fuel Oil (WAF). 


a> c 
-C ns 



141 


ppm ± s.d. (N measurements). 



100 - 



Figure 11-3. Batch variability in N. obsoletus larval 
survival upon exposure to "1.0" ppm fuel oil (WAF). 


NOTE: Experiment "A" employed 15 larvae/vial and ran 4 days. Experiment 
"B" employed 20 larvae/vial and ran 8 days. The range of mortality observed 
in the 3 replicates run at each concentration is indicated. Oil concentrations 
are reported as Mean ppm ± s.d. (N measurements). 


Static exposures of larval C. fornicata ran only three days. No mortality was 
observed at any oil level tested. However, no larvae were observed swimming at 
“1.0” ppm by day two. Activity was normal at lower oil concentrations. 
Moreover, the guts of larvae at “1.0” ppm were empty of food by the second 
day, whereas veligers at lower oil concentrations continued to feed during the 
three-day experiment. In the flow-through exposure, veligers held at “1.0” 
ppm also stopped swimming by the second day of the experiment. These larvae 
were alive but emaciated by day four, and dead by day six. Crepidnla fornicata 
larva had good survival at lower oil levels, although growth rates were affected 
as discussed below. 

Gastropod Reproduction 

Adults of N. obsoletus collected in February, 1976, and October, 1976, 
deposited their first egg capsules in laboratory control containers on 4/30/76 
and 5/6/77, respectively, when the water temperature warmed to 
approximately 10°C. Sastry (27) also obtained egg capsules at 10°C from N. 
obsoletus collected in January at Beaufort, North Carolina. 


142 













100 



Figure 11-4. Survival of C. irroratus during development 

at different oil concentrations. 


NOTE: Mean oil concentrations ± s.d. (N) were "0.01" ppm: 0.011 ppm 
±0.009 (14); "0.1" ppm: 0.094 ppm ±0.027 (14); "1.0" ppm: 0.913 ppm ± 
0.210 (14). 


Onset of egg capsule deposition by snails exposed to “0.01” ppm and “0.1” 
ppm was delayed by about two weeks relative to controls in 1976, and delayed 
up to one week in 1977. Encapsulated embryos produced by these oil-exposed 
snails, and transferred to control conditions, developed to hatching without 
noticeable abnormality. In a mid-summer experiment, control individuals 
produced many capsules within three days of collection from the field, but 
those held at “1.0” ppm never deposited any capsules. 

Egg capsule production is used here as an index of fecundity for both N. 
obsoletus and U. cinerea , since exposure to oil did not alter the average number 
of eggs per capsule (Table 11-2). 

Egg capsule production by N. obsoletus held at “0.1” ppm may be reduced 
relative to control, and “0.01” ppm snails (Table 11-3, line “e”), although 
these data are insufficient for statistical analysis. Results are inconclusive due 
to the death of an unknown number of females during the test breeding period, 
precluding accurate calculation of individual fecundity. 


143 












Table 11-2. Effect of Exposure to No. 2 Fuel Oil (WAF) on the Distribution 
of Eggs Among Egg Capsules of N. obsoletus and U. cinerea. 


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NOTE: F -values calculated by one-way analysis of variance. Oil concentration is given as Mean ppm ± 2.d. 
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NOTE. Each container initially held 35 snails. Two groups of snails were exposed at each oil level. Dates of exposure were 
2/13/76 — 8/9/76. Measured oil concentrations are given in Figure 11-5. 






Oil exposure modified the normal pattern of egg capsule deposition by adult 
N. obsoletus (Figures 11-5 and 11-6). Control snails tended to climb up the 
sides of the containers and deposit egg capsules mostly on the underside of the 
lid (Table 11-4). In the intertidal zone where spawning occurs, this behavior 
would contribute to placement of egg capsules into high-humidity 
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and “0.1” ppm oil deposited capsules primarily on the container sides (Table 
11-4). This effect was consistently most pronounced in May and June, at the 
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LlI 

Q 



Figure 11-5. influence of No. 2 fuel oil on egg capsule 
deposition behavior of N. obsoletus, 1976. 


NOTE: Exposures were initiated 2/13/76. Each point represents data pooled 
from 2-4 replicate containers holding fifty snails each. The total number of 
capsules deposited were: 19,492 (control); 9,894 ("0.01" ppm); 11,287 ("0.1" 
ppm), indicates the transfer of two control containers to "0.01" ppm. Mean 
total petroleum hydrocarbon concentrations ± s.d. (N) at each nominal con¬ 
centration were "0.01" ppm: 0.020 ±0.008 (9); "0.1" ppm: 0.082 ±0 044 
(20). 


146 







3ai s no sainsdvo on / an no sdinsdvo on 


Figure 11-6. Influence of No. 2 fuel oil on egg capsule 
deposition behavior of N. obsoletus, 1977. 


NOTE: Exposures initiated 10/22/76. Open circles represent data from con¬ 
trol snails, closed circles represent data from snails held at "0.1" ppm. Mean 
oil hydrocarbon concentration at "0.1" ppm ± s.d. (N) was 0.077 ppm ±0.026 
(34). Arrows indicate reversal of treatments. 


147 

















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148 






oil-exposed snails, with one exception (i.e., 6/10/76). After transfer of two 
control containers to “0.1” ppm, lid deposition decreased and remained 
suppressed for some 30 days (Figure 11-5). In a similar experiment conducted 
the next spring, control containers were transferred to “0.1” ppm and 
containers held at “0.1” ppm were transferred to control conditions. Shifts in 
deposition patterns were again observed (Figure 11-6), although not until after 
four weeks for the former control group of snails. 

With oyster drills ( Ucinerea ), there was no demonstrable effect of oil on 
fecundity when tested by one-way analysis of variance (P > 0.25), nor 
did exposure to “0.01” ppm and “0.1” ppm have any statistically significant 
effect on egg capsule placement. Egg capsule deposition behavior of oyster 
drills was affected by oil, however. No drills held at “0.1” ppm deposited 
capsules on the undersides of the container lids, in contrast to 13% to 14% lid 
deposition in 0.01 ppm and control treatments, respectively (Table 11-5). 

Larval Growth 

The influence of No. 2 fuel oil (WAF) on larval growth of three invertebrate 
species is summarized in Table 11-6. Growth of N. obsoletus larvae (jam shell 
length) was dramatically impaired at “0.01” ppm and “1.0” ppm, but was only 
slightly reduced at “0.1” ppm. This curious response pattern was observed in 
two experiments involving different hatches of larvae. In contrast, the effect of 
oil on larval growth of C. fornicata correlated positively with increasing oil 
concentration in the static experiments. Reduced growth was also evident at 
“0.1” ppm in the single flow-through experiment conducted. Growth of C. 
irroratus larvae, measured as change in mean dry weight/individual, declined 
relative to controls as development proceeded at the “0.1” ppm concentration. 
The dry weight of oil-exposed larvae was only 70.1 percent of control weight 
for the Stage IV zoea, and only 63 percent of control weight for the Stage V 
zoea. However, these same C. irroratus larvae exhibited no differences in 
molting frequency or in carapace length with respect to control individuals. 
Cancer irroratus larvae in all sublethal oil treatments (i.e., all below “1.0” ppm) 
reached the megalops stage in 25-28 days. 

DISCUSSION 

The concentration of hydrocarbons lethal to larvae is generally believed to 
be about one-tenth of the concentration lethal to adults (14, 21). In our study, 
a nominal concentration of 1.0 ppm was lethal to adults and larvae alike, 
although 50% mortality of larval N. obsoletus occurred much sooner for larvae 
than for adults. Exposure to “0.01” ppm and “0.1” ppm produced mainly 
sublethal effects in both larvae and adults of this species. 


149 


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Oil concentrations are given in Table 11-2. 







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concentration. 


Experiments on pollutant toxicity are usually conducted under constant 
conditions of light, temperature, and salinity. The results of such experiments 
may not be applicable to the field, where environmental conditions vary. A 
case in point is the observed seasonal variation in oil toxicity to adult N. 
obsoletus , with toxicity being accentuated in winter. Egg capsule deposition 
patterns of oil-exposed N. obsoletus also showed temporal variability. Patterns 
were least like those for control snails when water temperature was low at the 
beginning of the breeding season, but changed substantially as water 
temperatures rose. Similarly, Krebs and Burns (17) have observed that fiddler 
crabs (Uca pugnax ) exposed to No. 2 fuel oil in the field, show abnormal 
behavior only at temperatures near the lower limit of their normal range of 
activity. 

There are several possible explanations of the greater toxicity of this oil at 
low temperatures. Low temperature may directly increase the relative 
concentration of the more toxic oil fractions present in the water, either 
through altered solubility, volatization, or shifts in bacterial activity. This 
hypothesis is currently being explored. It is also possible that seasonal changes 
in toxicity result from changes in the physiological state of the animals and/or 
from additive effects of low temperature and oil stress. 

There is currently little information on how petroleum hydrocarbons enter 
aquatic animals, but recent evidence indicates that uptake of oil through 
ingestion of contaminated food may be at least as important as diffusional 
uptake (8, 11, 18). This is consistent with our observations of higher mortality 
of adult N. obsoletus in the presence of sediment. This occurred only during 
the summer, when N. obsoletus is actively deposit-feeding (30). The sediment 
effect did not occur during the winter, when the snails are inactive. 

We observed several sublethal effects on invertebrate reproduction, 
including possible reduction in the fecundity of N. obsoletus. Although there 
was no alteration in the number of eggs per capsule, the number of capsules 
produced appeared to decline. One possible variable influencing egg capsule 
production may be date of initiation of oil exposure relative to the onset of 
oogenesis. In this study, U. cinerea were exposed to hydrocarbons after 
gametogenesis was completed, and egg capsule depostion was already 
underway, which might explain the absence of a fecundity response for this 
species. More data are needed to resolve this issue. 

The observed alteration of egg capsule deposition behavior with respect to 
substrate orientation has significant ecological consequences for N. obsoletus. 
The egg capsules and embryos of N. obsoletus are not well adapted for 
deposition in the exposed intertidal zone; successful pre-hatching development 
of this species is apparently dependent instead upon the proper placement of 


152 


the capsules on substrates (23). Interference, with normal patterns of egg 
capsule deposition behavior could substantially increase pre-hatching mortality 
from desiccation stress (23). 


The reduction in larval growth rate observed at sublethal oil concentrations 
could result from increased energy expenditure, decreased ingestion rate, 
decreased assimilation efficiency, or a combination of these factors. Present 
evidence suggests that the reduced growth observed was due at least in part to 
reduced food intake. Larvae of C. fornicata and N. obsoletus held at “1.0” 
ppm ceased feeding at least one to two days before they died. The larval guts 
of C. fornicata were empty of food by the second day of each experiment, 
even though the velar lobes remained extended and ciliary activity was 
observed. Tissues in these individuals became dramatically shrunken within 
several days after initiation of exposure to oil. Veligers held at oil 
concentrations of “0.10” ppm showed no such morphological abnormality, but 
preliminary experiments (Pechenik, unpublished) reveal decreased ingestion 
rates at this concentration, relative to ingestion rates of control larvae. 


It is not yet possible to precisely predict the threshold oil concentrations at 
which lethal or sublethal effects occur. The potential for seasonal changes in oil 
toxicity has already been discussed. Moreover, most laboratory experiments 
conducted to date, including many in the present study, have used static 
exposures in which the dosing medium is replenished at one to two day 
intervals. Due to loss of volatile fractions from these aqueous mixtures, the 
initial hydrocarbon concentrations cited represent only maximum 
concentrations which the animals experienced during a test (6, 32). Atkinson 
et al (4) reported that 90 percent of the benzene initially present in a test 
solution is lost from undisturbed cotton-plugged flasks within a 24 hour 
period. Our containers were kept tightly sealed in the static experiments, 
minimizing such loss. Some loss of hydrocarbons through volatilization could 
have occurred during transfer of the medium from the flow-through tanks to 
the experimental containers, however. Finally, oil concentrations are generally 
reported as total hydrocarbon content, as measured by infrared 
spectrophotometry. Yet, toxicity to animals is probably due to only a small 
fraction of the hydrocarbon compounds present in the water accommodated 
fraction used (17), a fraction which can vary qualitatively and quantitatively 
over the period of an investigation. The concentration of specific oil fractions 
present during an experiment is generally unknown. Better control and analysis 
of oil exposures conditions are needed if we wish to accurately determine 
threshold concentrations of oil which are toxic to marine organisms. 


153 


ACKNOWLEDGEMENTS 


We wish to thank A. Ackenhusen, L. Halderman, E. Kenyon and N. Miller 
for their assistance in various aspects of this study. L. Halderman and S. 
Sosnowski provided helpful criticisms during manuscript preparation. The 
flow-through dosing system was maintained by R. Pruell and B. Melzian. 

4 

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13. Hargis, W.J., Jr. 1957. A Rapid Live-Sexing Technique for Urosalpinx 
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22. Nicol, J.A.C., W.H. Donahue, R.T. Wang, and K. Winters. 1977. Chemical 
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32. Wells, P.G. 1972. Influence of Venezuelan Crude Oil on Lobster Larvae. 
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33. Wells, P.G., and J.B. Sprague. 1976. Effects of Crude Oil on American 
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Meeresunters. 5: 169. 


156 


EXTRACTION OF ENVIRONMENTAL 
INFORMATION STORED 
IN MOLLUSCAN SHELLS: 
APPLICATION TO ECOLOGICAL PROBLEMS 


Donald C. Rhoads and Richard A. Lutz 
Department of Geology and Geophysics 

Yale University 

New Haven, Connecticut 06520 


ABSTRACT 

Ecological stress, when broadly defined, is responsible for most, if not all, 
growth patterns within the molluscan shell. As the type of pattern deposited is 
largely a function of the specific biological or environmental stress involved, 
considerable ecological information is stored within the exoskeleton. The 
resulting record is in the form of either (1) microstructural growth increment 
sequences or (2) changes in the shell structural type (e.g., nacreous, prismatic, 
crossed-lamellar, etc.) or relative proportions of structures within the shell. 

Microstructural growth increments, heretofore interpreted as resulting from 
varible despositional rates of calcium carbonate and organic matrix, are viewed 
as refections of periodic shell dissolution-deposition cycles. 

Changes in the type of crystalline structure deposited under various 
environmental conditions within the inner shell layer of several species of 
bivalves have been defined. During periods of extreme ecological stress, such as 
prolonged exposure to sub-freezing temperatures, extensive dissolution and 
“reworking” of this inner layer occurs in a number of species. 

Extraction of environmental information recorded within the shell is 
facilitated through examination of polished thin sections, acetate peels, 
fractured shells, polished and etched shell sections, and growth surfaces using 
polarizing, optical, and scanning (and, occasionally, transmission) electron 
microscopy. Application of these techniques to long-term monitoring of 
ecologically stressed environments is discussed. 

INTRODUCTION 

Ecology has been defined as the study of relationships between organisms 
and their environment (28). In functioning ecosystems it is possible to make 
direct observations of these relationships in real time. Organism-environment 


157 


relation ships may not always be directly measured, however, requiring an 
indirect or deductive approach; for instance, we may wish to assess the effect 
of a storm, pollution event, change in salinity, temperature, etc. on a species 
population after the event has taken place. In the absence of data about 
pre-disturbance rates of growth, death, and reproduction, we are totally 
dependent on indirect techniques. This kind of after-the-fact problem is 
common in paleoecology and promises to be an increasingly important 
approach in pollution biology*. 

Subject matter of the present article has been extracted and condensed from 
initial drafts of a manual which is currently being prepared for the 
Environmental Protection Agency * 2 3 4 . The purpose of this manual is to bring 
together and organize paleoecological literature so that it may be of use to the 
pollution biologist confronted with after-the-fact monitoring 2 problems. 

The Skeletal Record 

Skeletonized organisms provide an opportunity for deducing ecologic 
relationships in the past. The skeleton often contains a record of dynamic life 
and death processes, and provides both ontogenetic and demographic^ 
information. Ontogenetic data are related to the life history of an individual. 
Growth rates may be resolved to a high level of resolution from mineralized 
tissue showing growth banding correlated with lunar and/or solar cycles, or 
seasonal changes in water temperature, salinity, day length, primary 
productivity, etc. Biological events, such as season of reproduction and death, 
may also be recorded. Demographic data are related to population structure 
and its maintenance; growth, mortality, recruitment, and migration. The unit 
of study is a single species population. In the present article, we will limit our 
discussion to extraction of ontogenetic data. 


* Paleoecologic research over the past decade has developed many techniques for 
reconstructing paleoenviroments (30-32). Much of this literature is unknown to 
neontologists. 

2 

Preparation of the manual is supported by Environmental Protection Agency grant 
R 804-909-010. 

3 

The term monitoring is used here to describe reconstruction of an organism’s history 
of growth, reproduction, and mortality as preserved in its skeletal parts. Inferences 
about environmental causes for the observed record are, by definition, indirect and 
deductive. 

4 

Demography, taken literally, means writing about the people (Gr. demos, the people + 
to write). The term was originally used to describe statistical studies of human 
populations; births, deaths, marriages, etc. We use demography in a broader sense; the 
statistical description of populations of any taxonomic group. 


158 



Although all organisms with either an exoskeleton or endoskeleton can 
potentially provide ontogenetic data, bivalve molluscs are the most universally 
used group for obtaining these types of information for three reasons: 

(1) Most members of the Class Bivalvia are preservable, and are common 
faunal elements in both recent and fossil assemblages. Many species are 
present in areas impacted by pollution and are represented in both 
early and late stages of ecological successions following seafloor 
disturbance. 

(2) Preparation of the shell for obtaining ontogenetic information is easily 
done. This involves sectioning or fracturing the shell along a plane 
passing from the oldest part of the shell, the umbo, to the growing edge 
along the maximum axis of growth (30, 32). Coiled or otherwise 
torqued shells (e.g. gastropods) make this technique impossible with 
present methods. 

(3) Most research relating shell parameters to environmental conditions is 
based on bivalves. 

Data from Living and Dead Molluscs 

The relationship of a species to its environment has been conceptualized in 
the niche model (28). The species of interest is able to grow and reproduce as 
long as the organisms’ functional range (biospace) is not exceeded by the 
ambient environment. Not all parts of the realized biospace promote equal 
growth or fecundity. Different combinations of niche parameters will be 
manifested in changed rates of growth, survivorship, or reproductive success. 
All of these manifestations are capable of being preserved within the shell. We 
therefore have a record of an organism’s responses to changing niche conditions 
preserved in shells of individuals, composing either the living or death 
assemblage. 

Suboptimal niche conditions can be thought of as ecological stress. 
Ecological stress is responsible for most, if not all, growth patterns within 
individual shells. In this regard, an ecological stress, such as a pollution event, 
can be assigned dimensions in both space and time. These dimensions are 
important when considering species appropriate for establishing after-the-fact 
relationships. Ideally, the spatial distribution of a species should overlap and 
extend beyond the affected area. Populations falling outside of the polluted 
area can be used as control or reference populations. If the pollution event is 
lethal to part or all members of the population occupying the affected seafloor, 
after-the-fact study will include a comparison of both living and death 
assemblages. If the effect is sublethal, living assemblages alone will be used. 


159 


The duration of the pollution event should be considered relative to the 
mean life span of individuals (turnover rate). Again, the ideal situation is one 
where the species overlapping the affected area is one with a low turnover rate, 
and a life span that is long relative to the duration of the pollution event. If the 
ecological stress is sublethal, a record of growth before, during, and after the 
stress event, may be recorded within the living population, and can be 
compared with that of the reference population outside the affected area. If 
the stress results in high mortality, the death assemblage may be all that 
remains to document the event. 

MOLLUSCAN GROWTH PATTERNS 

Environmental information is stored within the molluscan shell in the form 
of either (1) microstructural growth increment sequences or (2) changes in the 
shell structural type (e.g. nacreous, prismatic, crossed-lamellar, etc.) or relative 
proportions of structures within the shell. These two distinct types ot records 
and their usefulness in ecological studies are discussed below. Much of this 
discussion is taken directly from a recent article by Lutz and Rhoads (26). 

Microstructural Growth Patterns 


During the past decade, numerous workers (2-4, 13, 14, 30-32) have 
described microstructural increments within the molluscan shell. Asa result of 
marked periodicity associated with many of these structures, they have proved 
useful in geophysical studies for defining changes in the earth’s rotational rate 
(3, 29-31), in ecological and paleoecological studies for assessing the effects of 
various biological and environmental stresses (9, 14, 18, 30, 32), and in 
archaeological studies for reconstructing migration patterns of prehistoric 
hunter-gatherers (6, 7, 19). When shells are viewed in cross-section (procedural 
details outlined in Methods section below), these microstructural patterns are 
seen as alternating bands of shell material ranging in thickness from 10° to 10" 
ju. 


Many, if not all, microstructural periodicity structures within the molluscan 
shell are a reflection of variations in the relative proportions of organic material 
(conchiolin) and calcium carbonate (aragonite or calcite). Alternation of 
calcium carbonate-rich layers and organic-rich regions or lines has been well 
documented for numerous recent and fossil species through detailed studies of 
shell thin sections, acetate peels, and polished and etched surfaces, employing 
polarizing, optical, and scanning electron microscopy (see Methods section). 
“Daily” growth increments have been reported by several workers (2, 13, 14, 
18, 30-32). These “daily” lineations were originally interpreted as reflections 
of solar time (13, 14, 17, 30, 31). Recent studies, however, have revealed a 
complex relationship between incremental growth, and lunar and solar cycles. 


160 


Although a one-to-one correspondence has not been established, the deposition 
of increments in bivalves is highly correlated with shell valve movements (27, 
30, 35, 36). As the valves of many species are generally closed during low tide, 
and open during high tide, a high positive correlation also exists between the 
number of increments and the number of tides to which an organism has been 
subjected. While valve-movement rhythmicity is generally most pronounced in 
intertidal individuals, subtidal specimens of at least one species (Mercenaria 
mercenaria) exhibit biological rhythms in relative harmony with the tidal cycle. 
There is general agreement among growth line workers that when the valves are 
open and the organism is actively pumping, a layer is deposited which is rich in 
calcium carbonate relative to adjacent shell material. The origin of alternating 
layers or lines relatively rich in organic content has recently been theorized by 
Lutz and Rhoads (26). The following few paragraphs summarize this theory 
which is based on recent studies of molluscan anaerobiosis and mechanisms of 
shell formation. 

During aerobic metabolism, molluscs deposit calcium carbonate, in the form 
of either aragonite or calcite, together with organic material, resulting in shell 
construction. Such metabolism is usually highly correlated with periods of 
active pumping, during high tide in well-oxygenated waters. As the 
concentration of dissolved oxygen falls, such as in the microenvironment 
created by the organism during periods of shell closure, anaerobic respiratory 
pathways are employed and levels of succinic acid (or other acidic 
end-products) within the extrapallial fluid rise. The acid produced is gradually 
neutralized by shell calcium carbonate, leading to increased levels of Ca ++ and 
succinate (or other end-products) within the extrapallial and mantle fluids (8). 
As a result of this decalcification, the ratio of relatively acid-insoluble organic 
material to calcium carbonate increases at the mantle-shell interface. One need 
not invoke the complication of increased concentration of organic material in a 
given volume, although a collapse of unsupported matrix structures or 
movement of the mantle as a compensatory response to the increased 
mantle-shell distance could result in increased concentrations of freed organic 
material in specific regions of the extrapallial fluid. With the return of 
oxygenated conditions and resumption of aerobic metabolism, and assuming 
shell deposition during this post-anaerobic period proceeds via a process similar 
to that occurring immediately prior to anaerobiosis, deposition of calcium 
carbonate and organic material within an area already containing organic 
material should result in an increase in the organic/ CaCO^ ratio within the 
specific shell region. The end-product of this process, from a strictly structural 
viewpoint, is one growth increment. 


161 





Methods 


Microstructural increments can be studied in thin sections of shell material, 
in acetate peel replicas of acid-etched shell sections, or under the scanning 
electron microscope (fractured or polished and etched shell sections). 

Acetate peels are the easiest and most rapid method of preparation for 
examination of most molluscan shells. The basic method of preparation, as 
outlined by Rhoads and Pannella (32), is as follows: 

Shells are embedded in a block of epoxy resin (e.g., Epon 815 resin with 
DTA hardener, 10:1 ratio, under vacuum; Miller-Stephenson Chemical 
Company, Danbury, Connecticut) to avoid shell fracture during sectioning. The 
plane of the cross-section passes from the umbo to the shell edge along the axis 
of maximum growth (30, 32). This cut is oriented so that growth increments 
intersect the plane of the section at right angles. The cut shell surface is 
polished sequentially with 350, 600, and, finally, 2600 or 3000 grade 
carborundum grits. The polished surface is then etched with 0.1 N HC1 for 
periods varying from a few seconds to a few minutes. Optimal etching time is 
related to shell structure, mineralogy, organic content, and state of 
preservation. It is recommended that a series of test etching times be carried 
out to determine optimum etching periods for a particular set of specimens. 

Etched shell surfaces are flooded with acetone, and a piece of sheet acetate 
is applied to the etched shell surface and weighted to avoid bubble formation. 
After the acetone (solvent) has evaporated (approximately 30 minutes), the 
acetate is removed from the shell and examined under the microscope (or used 
as a negative by placing directly in a photographic enlarger and printing). This 
technique yields excellent results for most species. 

Thin-sections are necessary for the examination of growth increments which 
are not structurally discontinuous, but instead recognizable only by dark and 
light color bands (32). For example, the growth increment boundaries in the 
deep-water species, Nucula cancellata and Calyptogena ponderosa , are 
indistinct and recognizable only by color variations of the bands, each band 
consisting of one dark and one light layer. The initial procedure for making 
thin-sections is the same as that for preparation of acetate peels, however, after 
the cut shell surface has been polished, it is glued to a glass slide using epoxy 
resin. The majority of the embedded shell and remaining embedding material is 
cut away using a diamond rock cutting saw, and the new exposed surface is 
polished sequentially until a 0.03 mm thick section of material is left on the 
slide. A cover slip is glued onto the newly polished surface using epoxy resin, 
and the material examined using optical or polarizing microscopy. Thin 
sectioning of shells is difficult, because shells tend to fracture when sectioned 


162 


and the micro-growth lines are obscured. In addition, it is difficult to avoid the 
formation of bubbles beneath the coverslip which obscure features. Equipment 
for preparing thin sections is available in rock preparation laboratories found in 
most geology departments. Alternatively, there are commercial firms (e.g., 
Rudolf von Huene, Pasadena, California) that will prepare thin sectioned 
material. 

Ecological Applications 

Several workers (9, 14, 18, 30-32) have suggested that information about 
physiological and environmental conditions may be recorded and stored in 
molluscan shells. Various studies in which workers have used microstructural 
increments within shells to extract such information are discussed below under 
appropriate sub-headings. 

Seasonal Cycles 

Seasonally caused annual growth rates and patterns are observable in all 
bivalves collected in climatic zones, ranging from cold-temperate to 
sub-tropical. In many species, as winter approaches, there is a gradual slowing 
down of the deposition rate, and the microstructural increments become 
gradually thinner. This slowing down of growth in the autumn culminates in a 
marked depositional break at the time of the first freeze. These depositional 
breaks are characterized by indentations of the outer shell layer, a dark band of 
organic-rich shell material extending downward from the base of the 
indentation, small daily growth increments on either side of the break, and a 
change in the shell structure near the break (9). These winter breaks may not 
be as marked in specimens living subtidally (32), although Mercenaria 
mercenaria from water depths of eight meters clearly show a distinct winter 
break (9). 

Through careful examination of microstructural patterns, Farrow (13) 
found that part of a population of the shallow sub tidal cockle, Cerastoderma 
edule, from the Thames estuary in England, stopped growing during winter due 
to sub-zero temperatures. Tevesz (34) observed that Gemma gemma grew very 
little in the winter. Growth increments were very closely spaced and the inner 
shell layer had a brownish hue. During the summer, G. gemma grew rapidly; 
microstructural increments were widely spaced, and the inner shell layer was 
clear and translucent in appearance. 

Through an examination of numerous acetate peels, Evans and LeMessurier 
(12) were able to demonstrate striking winter growth rate differences between 
two sympatric species of bivalves. They found winter growth of the 
rock-boring clam, Penitella penita, to be approximately 75 percent of the 


163 


summer growth rate, while growth of the cockle, Clinocardium nuttalli, which 
inhabited a neighboring mud flat, was slowed by as much as a factor of 19. 

In addition to rhythms based on periodic environmental fluctuations, 
biological rhythms, such as breeding periods, are also reflected in growth 
increment clustering. “Breaks” due to spawning events are less severe than 
winter breaks. They are preceded by little or no slowed growth, and recovery is 
more rapid than after winter breaks. In most bivalves, spawning occurs during 
the summer, sometimes more than once a year. In Mercenaria mercenaria , 
reproductive breaks do not occur until the second year of growth (32). 

Semiperiodic and Random Events 

Depositional breaks in bivalves also result from semiperiodic or random 
events such as storms, unseasonable temperatures, attacks by predators, and 
environmental pollution disturbances. The irregular nature of such events 
makes them easily separable from periodic or cyclical shell-secretion rhythms 
(18, 30, 32). 

Storm breaks, a common feature, may have different characteristics 
depending on the severity of the storm and depth at which the bivalves live 
below the water surface. In any case, these breaks appear suddenly and are 
followed by a rapid return to increments of pre-storm width (9). Shuster (33) 
noted that during storms, silt became trapped between the mantle and the shell 
in My a arenaria and was subsequently incorporated into the shell. Trapped silt 
within shell indentations formed by storm breaks has also been observed in 
Mercenaria mercenaria (9, 18). 


The Season, Age, and Frequency of Reproduction and Death 

Growth patterns can supply detailed information on the age of individuals 
at time of death and their season of death. The age at death will be: 


2 

where Ad is age at death and N s and N w are, respectively, the number of 
summer and winter bands in the shell (32). The season of death is determined 
by relating the position of the last increment at the margin of the shell to the 
seasonal growth pattern. For example, a margin preceded by a complete 
summer depositional record represents death in early fall. A margin which 
follows a long period of winter growth represents late winter or early spring 
death. Often, the shell margin is preceded by a few days of growth slowdown, 
and comparison of several individuals may be necessary in order to determine if 
the slowing down in growth represents a moribund condition prior to death, or 
is related to seasonal changes (32). By counting the number of increments in a 


164 



dead shell, it is also possible to relate the season of death to absolute age at 
death. 

The age at sexual maturity and season of reproduction can be determined 
by relating the position of spawning breaks to absolute age and seasonal 
pattern of growth. An illustration of the usefulness of growth patterns in 
determining age and season of reproduction is given by Rhoads and Pannella 
(32). They examined a population of Gemma gemma from an intertidal muddy 
sand flat on Long Island Sound. Summer growth patterns in G. gemma 
consisted of thick increments (7-25 fj.) and were readily distinguished from 
winter ones which were thin (1-3 ju). A period of decreased growth was seen in 
shell sections and was interpreted by them as reflecting reproductive events 
which occurred at the beginning of summer deposition. These thin increments, 
if related to spawning, should be associated with a spawning break in the shell 
margin. Rhoads and Pannella (32) determined that the periods of highest stress 
and mortality were different for juvenile and mature bivalves. Specimens 3.2 
mm (generally less than 6 months old) died with greatest frequency from 
summer to mid-autumn. Older individuals died primarily in late fall or early 
winter. 

Ontogenetic Records of Environmental Change. 

In addition to episodic and periodic events, variations in environmental 
parameters including food supply, the type of substratum, salinity, oxygen 
content, turbidity, agitation, temperature, and population density can 
influence growth of bivalves. Hallam (15) reviews these various environmental 
parameters as causes of stunting and dwarfing in living and fossil marine 
benthic invertebrates. Several studies conducted within the past few years have 
used microscopic growth increments within shells to define the effects of 
various environmental perturbations on bivalve growth. Rhoads and Pannella 
(32), for example, through careful examination of both acetate peels and thin 
sections, have demonstrated that examination of both acetate peels and thin 
sections, have demonstrated that Mercenaria mercenaria grows faster in sandy 
sediments than in mud when other variables are eliminated. Farrow (13) used 
microstructural growth increments within the shell of Cerastoderma edule to 
illustrate that dense populations of the cockles had a much shorter growing 
season than sparse populations. An inverse relationship between individual size 
and population density of cockles was also noted. In a subsequent study, 
Farrow (14) used growth increments within the outer shell layer of this species 
to demonstrate that individuals living high in the intertidal zone were stunted. 
The higher shore cockles were situated near the high water mark, and, 
consequently, were aerially exposed for several days during neap tides. 
Following neap tide deceleration, there was a resumption of vigorous growth. 
Many of the high intertidal cockles were some two-thirds the size of individuals 
lower in the intertidal zone, where growth was more continuous. 


165 


In a recent study, Kennish and Olsson (18) studied the effects of thermal 
discharges on the microstructural growth of Mercenaria mercenaria in Barnegat 
Bay, New Jersey. They found that clams from within a mile and a half of the 
mouth of Oyster Creek, which carries the heated effluent from the Oyster 
Creek Nuclear Power Plant, had a much higher number of breaks in their shells, 
thinner shells, and slower summer growth than did clams farther from the river. 
Counting the growth increments back from the shell margin, they determined 
that many of the breaks occurred concurrently with rapidly decreasing water 
temperatures, resulting fro,m abrupt shut-downs of the power plant, or rapidly 
increasing temperatures associated with abrupt renewal of plant operations. 
The growth rate of M. mercenaria generally increases with increasing 
temperatures and peaks between 20-24°C; Haskin (communicated to Kennish 
and Olsson) found decreased growth above 26°C. The thermal effluent raised 
the water temperature in areas around the mouth of Oyster Creek 3-5°C above 
ambient. Kennish and Olsson (18) also suggested that the thermal effluent may 
be adversely affecting physiological functions other than growth. At the station 
nearest the effluent, no spawning breaks were observed within the shells, while 
they were seen in specimens from all the control sites. 

Shell Structural Changes 

In addition to changes in patterns of microstructural growth increment 
sequences, changes in the type of crystalline structure deposited under various 
environmental conditions have been observed within the shells (particularly 
within the inner shell layers) of numerous species of bivalves. Dodd (10) 
described environmentally-controlled variation in the relative proportions of 
nacreous and calcitic prismatic structures within the innermost shell layers of 
Mytilus californianus . Lutz (23) found annual variation in the thickness of 
nacreous laminae within the inner shell layer of Mytilus edulis , and suggested 
that such variation might be growth rate and/or temperature dependent, with 
relatively fine laminae being formed with increased growth rate and rising 
temperatures in the late spring. Bryan (5) examined the effects of oil spill 
remover (detergents) on the shell of the intertidal gastropod, Nucella lapillus, 
following the Torrey Canyon spill in March of 1967. The addition of toxic 
detergent BP 1002 applied to the Kuwait crude oil spill was effective in 
temporarily sealing the shell edge by continuing the inner nacreous layer to the 
outer surface. Subsequent shell growth on thin nacre produced a growth mark 
and lines of weakness in the shell. Kennish and Olsson (18) observed 
transgressing regions of crossed-lamellar structure within the outer shell layer 
of Mercenaria mercenaria associated with shell deposition occurring during 
periods of extreme ecological stress (winter freezes, high summer temperatures, 
and thermal shocks from abrupt changes in operations of a nuclear power 
plant). Farrow (14) noted similar transgressing regions of crossed-lamellar 
structure within the outer layer of Cerastoderma edule associated with winter 


166 


growth, particularly in specimens sampled from high elevations (highly stressed 
environments) within the intertidal zone. 

Lutz and Rhoads (26) have recently presented evidence that structural 
changes within the shells of certain bivalve species may reflect periodic 
dissolution and “reworking” of primary depositional structures during periods 
of extreme environment stress. Here, alternating periods of aerobic and 
anaerobic metabolism provide the driving forces for shell deposition and 
dissolution, respectively. Parallel annually-formed sub-layers of nacre and 
simple aragonitic prisms (24, 25) within the inner shell layer of the Atlantic 
ribbed mussel, Geukensia demissa (Figure 12-1), for example, were interpreted 
as reflective of seasonal metabolic changes. In populations from Gulf of Maine 
waters, nacre deposition was restricted to the relatively warm months of the 
year (24, 25). During both the fall and spring, nacreous tablets on the inner 
shell layer growth surface became smaller and less regular, showing visible signs 
of erosion in the form of marked pitting and “hollow crystals”, as well as 
increased proportions of Fine-grained structures. Differential dissolution of 
calcium carbonate and organic material was also often observed at the inner 
layer growth surface during these seasons (Figure 12-2). During the colder 
months of the year (January — March, with water temperatures below 3°C), 
shell erosion became visible to the naked eye, the entire inner shell surface 
often presenting a chalky white appearance. Ultrastructurally, this surface 
appeared uniformly fine-grained or “homogeneous” (Figure 12-3). Similar 
visible erosion has been reported in Mercenaria mercenaria after long periods of 
valve closure (11). The ability of G. demissa to endure anaerobiosis for 
extended periods has been well documented (20, 22), as has the relative 
increased efficiency in this species of some of the citric acid cycle enzymes in 
an anaerobic direction (16). The observed shell erosion may well be a reflection 
of buffering of acid end-products from anaerobic metabolism during the colder 
months, when oxygen transport into the cells should theoretically be reduced 
relative to that occurring at higher temperatures (21). Wibur (37) has suggested 
that during periods of “adverse environmental conditions”, shell decalcification 
may predominate over growth. The often-seen gradation in fractured, as well as 
polished and etched, vertical shell sections of G. demissa nacreous laminae into 
finely grained structures (suggestive of massive erosion), instead of regular 
prisms, (Figure 12-4) tends to support this view. 

SUMMARY 

(1) Environmental and biological events are recorded in the molluscan shell 
in the form of small-scale growth increments and/or changes in shell 
structure. 


167 




Figure 12-1. Parallel annually-formed sub-layers of nacre and 
simple aragonitic prisms within the inner shell layer of the 
Atlantic ribbed mussel, Geukensia demissa. 


NOTE: (A) Scanning electron micrograph of vertical fracture surface x240. 
(B) Acetate peel of polished and etched longitudinal shell section x400. 


168 






Figure 12-2. Scanning electron micrographs of the inner 
shell layer growth surface of Geukensia demissa 
showing natural shell dissolution. 


NOTE: The differential solubility of calcium carbonate and organic matrices is 
apparent. Stereo pairs were taken with a 6° angular displacement between ex¬ 
posures. (A) x5000. (B) x20000. 


169 












.isgt 

ir 

m## ' :; %u s * 

%V«v* 'r\ 

f ' .* ISN 


" > -% * s 1r». C 


s-> *;f- #> 


1 - *•%. 

^ 4 :/ 

* > ' ^ 


* ^ 


'$&*,.,• r i* %. '* . 

f **'Qt : >' * . 


. It; 


J *£? 

dL i 
*%r£ 

,j A 



Figure 12-3. Scanning electron micrographs of the inner 
shell layer of Geukensia demissa showing fine-grained 
structures reflective of extensive shell dissolution during the colder 
months of the year (February sample). 

NOTE: Stereo pair was taken with a 6° angular displacement between expo¬ 
sures. x3000. 


170 




E 

o 


co 

V> 

</) 


03 
"U 

CO 

• mm 

<Si 

c 

03 

ZJ 
0) 

u. 

H— CO 

O 5 

0) « 

|.E 

CO CO 


</) 

L. 

CO 


0) 4_ 

SZ o 

" H- 

03 3 
CO 

■ MM 
0) 

I- 

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D 

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NOTE: (A) Scanning electron micrograph of vertical fracture surface showing 
gradation of nacreous tablets into fine-grained structures at top and gradation 
of prisms into nacre at bottom x2000. The most recently deposited crystals 
are at the bottom of the micrograph. (B) Acetate peel showing similar grada¬ 
tions of nacre into fine-grained structures and prisms into nacre x125. Again, 
the most recently deposited crystals are at the bottom of the micrograph. 


171 








(2) Alternating aerobic-anaerobic metabolic cycles are proposed as the 
physiologic mechanism for forming shell periodicity structures. Aerobic 
respiration is associated with shell calcification. Shell closure, 
accompanied by anaerobic metabolism, results in shell decalcification; 
acidic end-products are neutralized by dissolution of shell calcium 
carbonate. 

(3) Shell growth patterns can be easily studied by preparing shell thin 
sections or acetate peel replicas of acid-etched shell sections. Scanning 
electron microscopy of fracture or polished and etched shell sections 
can also be employed. 

(4) Patterns of growth increment sequences and shell structural changes are 
related to seasonal climatic cycles and, on shorter time scales, to lunar 
and solar periodicities. Semiperiodic or random events, such as storms, 
sedimentation events, or biological events (e.g., reproduction) are 
superimposed as “noise” on the geophysical cycles. Causal effects for 
this “noise” can be deduced by detailed studies of the growth record. 

(5) Shell growth patterns have proven useful in paleoecologic 
reconstructions. Detailed analysis of these patterns also promises to be 
an efficient manner in which to conduct after-the-fact or retrospective 
monitoring studies of pollution events. 

ACKNOWLEDGEMENTS 

Parts of this manuscript are a summary from a document prepared by Dr. 
Josephine Yingst; a contributor to the E.P.A. shell-growth manual. We thank 
A.S. Pooley, E. Tveter Gallagher, and A. Krishnagopalan for technical 
assistance with the scanning electron microscopy; W.C. Phelps for preparation 
of specimens; and W.K. Sacco for his assistance in photograph reproductions. 
This research was supported in part by Environmental Protection Agency grant 
R804-909-010 and NOAA grants 04-6-158-44056, SGI-77-17, and 

04-7-158-44034. Contribution number 108 from the Ira C. Darling Center, 
University of Maine, Walpole, Maine, 04573. 


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172 


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6. Coutts, P. J. F. 1970. Bivalve Growth Patterning as a Method of Seasonal 
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8. Crenshaw, M. A. and J. M. Neff. 1969. Decalcification at the Mantle-Shell 
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9. Cunliffe, J. E. and M. J. Kennish. 1974. Shell Growth Patterns in the 
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11. Dugal, L. P. 1939. The Use of Calcareous Shell to Buffer the Product of 
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13. Farrow, G. E. 1971. Periodicity Structures in the Bivalve Shell: 
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14. . 1972. Periodicity Structures in the Bivalve Shell: Analysis of 
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173 


15. Hallam, A. 1965. Environmental Causes of Stunting in Living and Fossil 
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16. Hammen, C. S. and S. C. Lum. 1966. Fumarate Reductase and Succinate 
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17. House, M. R. and G. E. Farrow. 1968. Daily Growth Banding in the Shell 
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18. Kennish, M. J. and R. K. Olsson. 1975. Effects of Thermal Discharges on 
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19. Koike, H. 1973. Daily Growth Lines of the Clam Meretrix lusoria: A Basic 
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20. Kuenzler, E. J. 1961. Structure and Energy Flow of a Mussel Population in 
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21. Lange, R., H. Staaland and A. Mostad. 1972. The Effect of Salinity and 
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22. Lent, C. M. 1968. Air-Gaping by the Ribbed Mussel, Modiolus demissus 
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24. . 1976. Geographical and Seasonal Variation of the Shell Structure 
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25. . Annual Structural Changes in the Inner Shell Layer of Geukensia 
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26. and D. C. Rhoads. Anaerobiosis and a Theory of Growth Line 
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27. MacClintock, C. and G. Pannella. 1969. Time of Calcification in the Bivalve 
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174 


28. Odum, E. P. 1971. In: Fundamentals of Ecology, W. B. Saunders Co., 
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29. Pannella, G. 1976. Tidal Growth Patterns in Recent and Fossil Mollusc 
Bivalve Shells: a Tool for the Reconstruction of Paleotides. 
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30. and C. MacClintock. 1968. Biological and Environmental Rhythms 
Reflected in Molluscan Shell Growth. J. Paleontol. 42, No. 5 (Suppl.): 64. 

31. and M. N. Thompson. 1968. Paleontological Evidence of Variations 
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32. Rhoads, D. C. and G. Pannella. 1970. The Use of Molluscan Shell Growth 
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33. Shuster, C. N. and B. H. Pringle. 1969. Trace Metal Accumulation by the 
American Eastern Oyster Crassostrea virginica. Proc. Nat. Shellfish. Ass. 
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34. Tevesz, M. J. S. 1972. Implications of Absolute Age and Season of Death 
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35. Thompson, I. 1975. Biological Clocks and Shell Growth in Bivalves. In: 
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D. and Runcorn, S. K. (eds.), Wiley, London, U. K. pp. 149-161. 

36. Whyte, M. A. 1975. Time, Tide and the Cockle. In: Growth Rhythms and 
the History of the Earth’s Rotation, Rosenberg, G. D. and Runcorn, S. K. 
(eds.), Wiley, London, U. K. pp. 177-189. 

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103-145. 


175 


LABORATORY CULTURE OF 
MARINE FISH LARVAE 
AND THEIR ROLE IN 
MARINE ENVIRONMENTAL RESEARCH 


E.D. Houde and A. K. Taniguchi 
Rosenstiel School of Marine 
and Atmospheric Science 
University of Miami 
Miami, Florida 


ABSTRACT 

The capability to predictably culture marine fish larvae beyond embryonic 
and yolk-sac stages has been developed during the past 15 years. This has led to 
advances in our understanding of how environmental variables affect survival 
and eventual recruitment of fishes. Most marine fish larvae are planktonic 
carnivores and consume living prey less than 150 jum in breadth when they first 
feed. The most important advance in culture technology was the determination 
of kinds and concentrations of prey that enable larvae to survive and grow at 
predictable rates, permitting ecological, physiological, and behavioral research 
to be undertaken. Prey concentrations necessary for growth and survival of 
some typical marine teleost larvae, usually range from 10^ to 1CP per liter. 
Best survival rates, fastest growth, and lowest variability, have been obtained at 
the 1CF per liter concentration. Growth efficiencies and food consumption by 
marine fish larvae are comparable to other predatory zooplankton. Some 
knowledge about effects of predation on marine fish larvae survival has been 
obtained, but further study is necessary, especially to determine how 
environmental factors modify predator effects. Some areas of environmental 
research, using cultured marine fish larvae, are reviewed. These include the 
roles of physical and chemical variables, other than man-induced environmental 
perturbations, and some effects of environmental change caused by man’s 
encroachment upon and alteration of marine habitats. Other important 
advances include development of field bioassay methods to determine if 
plankton standing stock can support fish larvae; development of biochemical 
and histological techniques to evaluate larval condition; and the recent 
discovery that larvae can be accurately aged using daily otolith increments. 
Some ideas for productive future research are proposed. 


176 


INTRODUCTION 


Fishes are large and conspicuous members of marine communities. They 
have important commercial and recreational value, and their abundance can 
fluctuate widely in response to environmental variability or heavy exploitation. 
Fluctuations in abundance of fishes usually are caused by large annual 
differences in recruitment, which are related to mortality experienced by a 
cohort during the larval stage (28, 29). It has been difficult to evaluate 
potential factors that could affect recruitment in studies carried out on natural 
populations at sea because of the problem in estimating egg or larval 
abundances over extensive ocean areas, and because of an unpredictable 
environment whose effects cannot be controlled. During the past 15 years a 
capability has been developed by several laboratories to routinely culture 
marine fish larvae beyond embryonic and yolk-sac stages to the juvenile stage. 
Experiments on these laboratory cultured species has resulted in significant 
advances in our understanding of how environmental factors affect survival and 
growth of larvae. 

Several papers recently have reviewed aspects of marine fish larvae culture 
(42, 50, 64, 65, 67). Although May’s (67) evaluation of the critical period 
hypothesis, and Iwai’s (50) review of feeding by fish larvae, included 
discussions of both laboratory and field-oriented studies, they did not 
specifically make conclusions about larval requirements based on experimental 
research. Blaxter’s (14) general review of egg and larval development of fishes 
did summarize results of laboratory studies. We review some important results 
of recent experimental research on marine fish larvae and make conclusions 
about effects of environmental factors based on laboratory studies. Emphasis is 
on studies of species that have typical, pelagic larvae and includes the period 
from initiation of feeding until transformation to juvenile. Research on embryo 
and yolk-sac stages, aquaculture-oriented studies, and work on non-pelagic or 
non-typical larvae are not emphasized, although important contributions have 
been made in recent years. 

Two major areas of research are reviewed and discussed. These are 1) the 
role of the food supply, the ability to feed, and the effects of predators; and 2) 
the role of physical and chemical variables, other than those due to man’s 
impact on the environment. In addition, new techniques that hold promise for 
advancing environmental research on fish larvae are outlined and discussed. 

FOOD REQUIREMENTS 

The most important advance in larval culture technology during the past 1 5 
years has been the determination of kinds and concentrations of living prey 
that give predictable survival and growth rates. The ability to undertake 


177 


meaningful ecological, physiological and behavioral research developed once 
larvae could be routinely cultured. A myriad of foods has been used to rear 
marine fish larvae (64), but five foods have been more successful in recent 
years for meeting larval nutritional requirements. These are the rotifer 
Brachionus plicatilis, the nauplius of brine shrimp Artemia salina , copepods 
from wild plankton collections, the harpacticoid copepods Tisbe and Tigriopus 
spp., and the naked di no flagellate Gymnodinium splendens. 

Prey Concentrations 

Marine fish larvae are visual feeders, with limited ability to search a volume 
of water for suitable food items during a unit of time. Suitable items usually 
are living organisms of a size that can be ingested, are nutritionally adequate, 
and are present at concentrations which allow a larva to encounter enough 
items during a day to meet its metabolic demands and to provide some excess 
for growth. Typical marine fish larvae are 2-3.5 mm long when they begin to 
search actively for food. Acceptable prey usually are 20-150 jum in breadth (7, 
31, 56, 92). Some large and rather atypical larvae, like Atlantic herring, Clupea 
harengus , or plaice Pleuronectes platessa, can begin feeding on items in excess 
of 300 jum in breadth (10, 80, 82). Perhaps not surprisingly, required 
concentrations of prey for newly-feeding larvae have been shown to vary 
greatly in laboratory studies, the variation in large part reflecting size 
differences in the prey that has been offered. 


Prey concentrations that have been used successfully to rear larvae have 
ranged from 1 x 10^ to 2 x 10^ per liter, although required concentrations for 
significant survival probably lie in the range 10^ to 10^ per liter. The highest 
reported concentrations (1-2 x 10^ per liter) were of the large dinoflagellate 
Gymnodinium splendens , which can be used to culture northern anchovy 
larvae during the first week of life (47, 57, 95). Lowest concentrations (442 
per liter) were of brine shrimp Artemia salina nauplii used to culture Atlantic 
herring larvae (82, 83). Neither G. splendens nor A. salina is usually available to 
marine fish larvae in nature, although Kiefer and Lasker (53) recently have 
shown that G. splendens may be present at 14 x 1(L per liter in the 
chlorophyll maximum layer of the Southern California Bight. Northern 
anchovy larvae can concentrate in the chlorophyll maximum layer and can feed 
on G. splendens when its concentration exceeds 2x10^ per liter (56). The 
most common prey reported from stomach analyses of marine fish larvae in 
nature are nauplii and other stages of copepods. Using copepod nauplii as food 
Houde (45) reported 10 percent survival at metamorphosis when per liter 
nauplii concentrations were 34 for sea bream Archosargus rhomboidalis, 107 
for bay anchovy Anchoa mitchilli, and 130 for lined sole, Achirus lineatus. 
Other studies with wild plankton (predominantly copepod nauplii) as prey 
have reported higher concentrations required for significant survival than those 


178 




reported by Houde (45). O’Connell and Raymond (73) estimated that more 
than 1000 nauplii per liter were required by northern anchovy larvae. Haddock 
Melanogrammus aeglefinus , larvae required 500-3000 per liter (58) and winter 
flounder, Pseudopleuronectes americanus , required 300-3000 per liter (60). It 
is possible that some reported prey concentrations required by larvae could be 
too high. Saksena and Houde (84) needed 1500-2000 nauplii per liter to 
successfully rear about 10 percent of bay anchovy larvae, but more recent 
experiments, with refined culture methods (45), have demonstrated that only 
100 nauplii per liter are necessary to attain that survival rate. In some research, 
such as toxicological studies to determine effects of pollutants on larval 
survival, potential survival rates higher than 10 percent are required. For those 
studies, copepod nauplii concentrations of 1000 per liter or higher should be 
routinely employed (44, 45) (Table 13-1). 

For six cases where copepod nauplii were fed to similar-sized larvae, the 
relationship between percent survival and nauplii concentration can be 
compared (Table 13-1). Haddock larvae had the highest required prey 
concentration, more than 2000 nauplii per liter being required for 10 percent 
survival. Winter flounder and northern anchovy larvae had an expected survival 
of 10 percent when nauplii were available at approximately 1600 and 1000 per 
liter, respectively. But, bay anchovy and lined sole required only about 100 
nauplii per liter and sea bream needed less than 50 per liter to attain 10 percent 
survival. All of these species consume prey of similar types and sizes; the 
differences in requirements among species have not been explained. 
Temperature may play a role because the three species with lowest required 
prey concentrations were reared at 26-28°C, while the three with higher 
requirements were reared at 7-17°C. If searching ability and capture efficiency 
are enhanced at higher temperatures, required prey concentrations may 
decrease accordingly. 

Rotifers, Brachionus plicatilis, are often used at high densities by 
aquaculturists to successfully rear fish larvae, but the minimum concentration 
required by larvae usually has not been determined. Hunter (46) estimated that 
105 rotifers per liter were required by newly-feeding northern anchovy larvae 
to meet metabolic demands, a number that must be exceeded for larvae to 
grow. Lined sole larvae required from 60-120 rotifers per liter for 10 percent 
survival to metamorphosis (Houde, unpublished data). 

Concentrations of microzooplankton in marine waters are not frequently 
reported, but when suitable collection techniques have been used observed, 
concentrations often are in the ranges of required prey densities determined in 
the laboratory (Table 13-2). Concentrations of suitable prey are exceptionally 
low in oceanic waters compared to coastal waters, and larval survival may 
depend upon the occurrence of relatively dense prey patches in oceanic areas. 


179 


Table 13-1. Copepod Nauplii Concentrations Used as Prey 
to Rear Six Species of Marine Fish Larvae and 
Corresponding Percent Survivals. 


Reference 

Species 

Stage to which 
larvae 

were reared 

Temperature 

(°C) 

Nauplii 
Concentra¬ 
tion 
(no./I) 

Percent 

Survival 

O'Connell and 

Northern anchovy 

12 days after 

17 

10 

0.0 

Raymond (1970) 

(Engraulis mordax) 

hatching 


100 

0.5 





1000 

12.0 





4000 

56.0 





8000 

25.0 





14000 

30.0 

Laurence (1974) 

Haddock 

metamorphosis 

7 

10 

0.0 


(Melanogrammus aeglefinus) 



100 

0.0 





500 

1.1 





1000 

7.9 





3000 

13.9 

Laurence (1977) 

Winter flounder 

metamorphosis 

8 

10 

0.0 


(Pseudopleuronectes americanus) 



100 

0.0 





500 

2.6 





1000 

3.8 





3000 

34.2 

Houde (in press) 

Bay anchovy 

metamorphosing 

26 

50 

11.6 


(Anchoa mitchilli) 



100 

4.7 





1000 

48.2 





5000 

63.9 

Houde (in press) 

Sea bream 

metamorphosis 

26 

10 

3.9 


(Archosargus rhomboidalis) 



25 

7.3 





50 

12.7 





100 

37.7 





500 

72.4 

Houde (in press) 

Lined sole 

nearly 

28 

50 

1.4 


(Achirus lineatus) 

metamorphosed 


100 

13.3 





1000 

54.3 


180 











Table 13-2. Reported Concentrations of Some Microzooplankton Suitable as Prey for 

Marine Fish Larvae from Coastal and Estuarine Areas. 




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181 











Even , in rich coastal waters, daily variability in microzooplankton 
concentrations occurs over order of magnitude ranges. Laboratory studies have 
shown that larvae deprived of food pass a “point of no return,” after which 
they cannot initiate feeding (21,67). This point can occur at only 0.5-2.5 days 
after yolk absorption for species at 20-32° (43, 57). Thus, unstable conditions 
that lead to temporary low prey concentrations probably are an important 
cause of mortality, even in areas where mean prey levels are high enough to 
sustain larvae. 

Growth of larvae in relation to prey concentration can be determined in the 
laboratory. There are, of course, factors other than density of prey which 
influence larval growth. The size of prey, their caloric value, their percentage 
protein, and their digestibility are important. The effect of temperature makes 
it difficult to compare growth among species of larvae, even when similar foods 
have been used. Despite limitations in the comparative approach, larval growth 
responses to changes in food concentration can be demonstrated in the 
laboratory, and results extended to explain how densities of prey influence 
growth of wild populations. 

A relationship between size at 16 days after hatching and copepod nauplii 
concentration was demonstrated for larvae of bay anchovy, lined sole, and sea 
bream (44, 45). Lengths and mean dry weights of survivors increased rapidly 
when prey level was raised from approximately 50 to 500 nauplii per liter. 
Lengths and weights tended toward asymptotes at food levels higher than 1000 
per liter, although significant, additional growth could be obtained at higher 
prey levels. Laurence’s data (58) on haddock larvae at six weeks of age show a 
similar relationship for prey concentrations in the range 500-3000 copepod 
nauplii per liter. Weights of winter flounder at 7 weeks of age in relation to 
copepod nauplii concentration also approached an asymptote at 1000 per liter 
prey level (60). O’Connell and Raymond (73) also found this type of 
relationship between length of northern anchovy larvae at 12 days and 
copepod nauplii concentration, except that prey ranged from 1000-14,000 
nauplii per liter and the asymptotic size was not attained until prey level was 
approximately 8000 nauplii per liter. 

Specific growth rates of marine fish larvae relative to prey concentration 
have been obtained in only a few instances. Specific growth rates (in dry 
weight) of haddock larvae were 7 percent, 8 percent, and 9 percent per day at 
copepod nauplii concentrations of 500, 1000 and 3000 per liter (58). The rates 
for winter flounder larvae, at the same nauplii concentrations were similar, 6 
percent, 8 percent and 9 percent (60). Temperature for haddock experiments 
was 7°C and for winter flounder it was 8°C. Specific growth rates of sea bream 
and bay anchovy larvae at 26°, and lined sole larvae at 28°C can be estimated 
from Houde’s data (45). The rates were 16, 20, and 28 percent per day for sea 


182 


bream at 50, 100 and 500 per liter copepod nauplii concentrations; they were 
16, 17 and 25 percent per day for bay anchovy at 50, 100 and 1000 per liter 
nauplii concentrations; but, lower rates of 7, 9 and 17 percent were obtained 
for lined soles at 50, 100 and 1000 per liter nauplii concentrations. Depending 
on prey concentration, the length of the larval stage can be highly variable. In 
the case of sea bream, specific growth rates at 100 and 50 per liter nauplii 
concentrations indicate that duration of the larval stage at those prey levels 
could be 1.4 to 1.7 times as long as at the 500 per liter level. Even if starvation 
was not a direct cause of mortahty at low prey levels, the indirect effects of 
increased time of exposure to predators and possible enviornmental stresses 
during the larval stage, must have important consequences on the numbers that 
eventually metamorphose. 

The density of prey, expressed as numbers per liter, provides a useful 
measure of availability of prey for capture by larvae, but does not necessarily 
provide a measure of energy available for growth and metabolism. Energy 
available is a function of prey density, prey size, and the ability of larvae to 
ingest particular prey, which is related to mouth size in many instances (10, 
90). The kinds of prey also could influence the availability of energy, either 
through differential ability of prey to escape capture by larvae, or through 
differences in caloric content of prey. Few studies concerned with marine fish 
larvae have taken a bioenergetic approach to examine nutritional requirements. 
Such studies can provide the means to estimate amounts of ingested energy 
used for growth and metabolism. Estimates of required food intake, specific 
ration, growth efficiency and the critical minimum prey level all can be 
determined on a caloric basis using this method. When used in conjunction 
with studies on feeding by larvae in relative to prey concentration, valuable 
insight into nutritional requirements and feeding dynamics can be obtained. 
Recent work by Laurence (60) on winter flounder larvae is the best example of 
the use of a bioenergetic model for marine fish larvae. 

The winter flounder larvae model (60) predicted critical food 
concentrations in the range 2.1-5.7 cal per liter, corresponding to 300-800 
copepod nauplii per liter. Highest prey concentrations were required by 
newly-feeding larvae, suggesting that food was most critical at that time. 
Smallest larvae required most of the daylight period to obtain a minimum 
ration. Relatively high metabolic energy demands were made by the smallest 
larvae, reflecting their low efficiency in capturing food. Metabolic demands 
were lowest at high prey concentrations because larvae expended less energy in 
searching when food was readily available. Thus, for winter flounder larvae it 
appears that food consumption needs to be higher at low prey concentrations 
than at high prey concentrations. Estimated minimum consumption ranged 
from 18-230 nauplii per day over a range of larval dry weights from 10-1000 
jug. Specific rations (jug consumed per jug larva x 100) decreased from nearly 


183 


300 percent for newly-feeding larvae to 27-31 percent tor the smallest larval 
stages (10-75 jug), and continued to increase slowly for older larvae, ranging 
from about 18-33 percent for metamorphosed individuals. Laurence (60) 
predicted a continuous decrease in growth efficiency as prey concentrations 
were decreased, but so few values of growth efficiency are available for fish 
larvae that it is not possible to say whether this relationship will hold for other 
species. 

Some valuable insight into feeding by marine fish larvae recently has been 
gained by combining results of bioenergetic studies on larvae with studies on 
feeding behavior and feeding ability of larvae. Blaxter and Staines (23) 
estimated swimming ability and feeding efficiency of herring, plaice, pilchard 
Sardina pilchardus , and sole Solea solea larvae. From their estimates they 
calculated the volume of water that could be effectively searched by larvae 
when they initiated feeding, and at sizes up to metamorphosis. Because 
swimming distances and volumes searched per unit time increased rapidly as 
larvae grew, larvae presumably needed higher prey concentrations during the 
youngest feeding stages. A similar approach was used by Rosenthal and Hempel 
(82), who in addition estimated the digestion time for herring larvae. They 
were then able to calculate the daily ration and required densities of prey 
(Artemia nauplii) for herring larvae at the end of the yolk-sac stage (10-11 mm) 
and at 13-14 mm length. Estimated ration was 40 Artemia nauplii per day at 
10-11 mm and 50 per day at 13-14 mm. Required Artemia concentrations for 
larvae to obtain the rations at each of those length-classes were 4 to 42, and 2 
to 25 per liter, respectively. Hunter (46) further extended the method by 
incorporating metabolic demands of larvae, and caloric values of prey (the 
rotifer Brachionus plicatilis and the dinoflagellate Gymnodinium splendens) 
into the prediction of food requirements. He concluded that first feeding 
northern anchovy larvae required 105 rotifers per liter or their caloric 
equivalents (e.g. 1785 Gymnodinium per liter) to just meet metabolic 
demands. Larvae at 10 days (5.9 mm) required only 34 rotifers per liter. In all 
of the examples, the relatively poor swimming ability and the low prey capture 
efficiency of first feeding larvae were demonstrated. This implies, as did 
Laurence’s study (60), that food concentration is most critical at the first 
feeding stage and, when low, could be a significant cause of larval mortality in 
the sea. 

It is possible to make many conclusions about larval food requirements 
based on dry weights of larvae, dry weights of prey, prey selection by larvae, 
digestion time, and estimates of the caloric values of the prey (cal/g ash free). 
Using these methods, Stepien (92) showed how feeding rates, specific rations 
and growth efficiency of sea bream larvae varied in relation to larval age, and to 
temperature for a single prey concentration. At 1000 copepod nauplii per liter, 
feeding rates for first feeding larvae (2-3 days after hatching) varied from 7.2 


184 


nauplii/hr/larva at 23°C to 17.6 nauplii/hr/larva at 29°C. These rates increased 
exponentially as larvae grew, so that larvae were consuming 53.8 
nauplii/hr/larva at 23°C and 142.7 nauplii/hr/larva at 29°C at 16 days of age. 
Rations, in terms of numbers of nauplii and dry weight consumption, were 
then calculated. Specific ration also was calculated, and it tended to decrease as 
larvae grew, particularly at the highest temperature (29°C), where it was 220.8 
percent at two days after hatching but decreased to 79.7 percent at 8 days. 
This result is similar to that of Laurence (60) for winter flounder (8°C), where 
specific ration decreased from over 300 percent for the smallest larvae to about 
30 percent for metamorphosed individuals. Mean gross growth efficiency of sea 
bream (92) varied from 23.9 to 30.6 percent, the highest value being obtained 
to the lowest temperature (23°C); there was no evidence that growth 
efficiency changed with age. Mean growth efficiencies were similar to those for 
winter flounder (60), except that first feeding winter flounder had low growth 
efficiencies, which increased rapidly during the first few days of active feeding. 
The relatively high growth efficiency of sea bream, when it begins to feed, 
suggests that food is less critical for it than for winter flounder at that stage, a 
suggestion supported by the relatively low required prey concentration for 
survival of sea bream larvae (45). 

Starvation Criteria 

Because starvation is suspected as a major cause of larval mortality, 
biochemical, histological, and behavioral criteria have been developed for some 
species to show changes that occur when the food supply is inadequate. These 
techniques eventually may be used to characterize starving larvae collected at 
sea. Biochemical methods also have been used to show how laboratory-reared 
larvae differ from wild larvae. When supported by morphometric data, 
biochemical criteria hold promise to evaluate how types and amounts of food 
affect larval condition. 

The biochemical composition of laboratory-reared, larval Atlantic herring 
and plaice was studied by Ehrlich (32, 33). He found that water, triglyceride, 
carbohydrate, nitrogen, carbon and ash content varied as a percentage of body 
weight as larvae grew, In starving larvae both relative (percentage) and absolute 
changes in amounts of those substances were measured, the relative changes 
often being a better measure of starvation than the absolute changes. 
Percentage of water increased about four percent in starved larvae of both 
herring and plaice, while percentages of triglyceride, carbohydrate, and carbon 
decreased. Percent nitrogen decreased in starved plaice larvae but did not 
decrease in herring; absolute amounts of nitrogen decreased in both species. 
Ash percentage of both species increased rapidly during starvation. Ehrlich (32, 
33) concluded that the “point of no return” was not defined by an abrupt 
change in the chemical composition at some point during starvation, but rather 


185 


that a continual change in chemistry occurred until the larvae became 
moribund. 

In a similar study Anraku and Azeta (6) compared cultured and wild larvae 
and juveniles of the sea bream Chrysophrys major. They did not examine larvae 
less than 10 mm length, but for large specimens the cultured individuals tended 
to have a lower percentage of water, higher percentages of carbon and 
hydrogen, and a percentage nitrogen that did not differ from wild specimens 
until 20 mm length, when percentage nitrogen decreased in cultured 
individuals. Differences in food of cultured and wild specimens were the 
probable cause of differences in body chemistry. Starved individuals of sea 
bream showed effects similar to those for herring and plaice — i.e. increased 
percentage water and decreased percentages of carbon, nitrogen and hydrogen. 

Histological changes in laboratory-reared larvae are indicative of starvation. 
Recent studies indicate that these criteria could be used to recognize starving 
or poorly nourished larvae in the sea. Umeda and Ochiai (96) examined fed and 
starved yellowtail Seriola quinqueradiata larvae, Ehrlich et al (34) examined 
herring and plaice larvae, and O’Connell (72) examined northern anchovy 
larvae. In all of these studies there were some similar findings. Intestinal 
epithelial cells atrophied in starving larvae and the intestine degenerated. The 
liver also degenerated in yellowtail, plaice and northern anchovy. O’Connell 
(72) and Umeda and Ochiai (96) examined the pancreas and found that its 
condition was markedly deteriorated in starved anchovy and yellowtail larvae. 
O’Connell (72) examined several other histological characters and found that 
starved anchovy larvae also had separations of muscular fibers and little 
intermuscular tissue, as well as notochord shrinkage. Using a discriminant 
function analysis he was able to discriminate 90 percent of starving larvae from 
fed larvae when four or more good histological characters were used. Ehrlich et 
al (34) found that there were good morphological characters associated with 
the histological changes, especially in herring larvae where severe head 
shrinkage and gut shrinkage caused a decrease in the “pectoral angle”, and an 
increase in the eye height to head height ratio. Histological criteria as indicators 
of impending starvation seem excellent. They are relatively time consuming 
compared to morphometric analyses, but perhaps are more effective to 
distinguish starvation effects. 

The concentration of prey affects larval behavior. Wyatt (100) 
demonstrated that duration of plaice larvae activity (searching behavior) was 
inversely related to prey concentration, and that starving larvae increased their 
time spent searching for food. This behavior presumably is adaptive and 
increases the probability of encountering prey when it is scarce. Using vertical 
migration as an index of activity, Blaxter and Ehrlich (20) found that fewer 
herring and plaice larvae vertically migrated after periods of starvation, and 


186 


that starved larvae tended to be neutrally buoyant because of a relative increase 
in body water. They speculated that under starvation conditions larvae would 
be relatively inactive, suspended in midwater, and thus more susceptible to 
plankton net sampling than well nourished larvae. Blaxter and Ehrlich’s (20) 
results differ somewhat from Wyatt’s (100), partly because of the different 
criteria used to define activity. 

Behavior of northern anchovy larvae in dense patches of prey, 
Gymnodinium splendens and Brachionus plicatilis, was investigated by Hunter 
and Thomas (49). In dense patches larvae swam slower and covered smaller 
areas. Reversals in swimming direction occurred more frequently in patches of 
food than in non-patch situations. The evidence strongly suggested that 
northern anchovy larvae were able to maintain themselves in suitable patches 
of prey, and that such an adaptation would allow larvae to take advantage of 
prey patchiness in the sea. 

PREDATION 

Predation almost certainly is the greatest direct cause of mortality to marine 
fish larvae, but there have been few attempts to evaluate its impact in 
laboratory studies. The food supply of larval fishes and other environmental 
factors can modify the predation mortality experienced by a cohort. As 
Cushing (29) noted, larvae that have an adequate food supply grow fast and 
swim well. Thus, they presumably avoid predation by growing quickly through 
the larval phase, when they are most vulnerable to a variety of planktonic 
predators. Similarly, pollutants or toxicants that retard larval growth or modify 
behavior could lead indirectly to increased predation mortality. 

Four recent laboratory investigations have examined the potential impact of 
predators on larvae. Three species of pontellid copepods could more than meet 
their metabolic requirements by preying upon yolk-sac larvae of northern 
anchovy (62). Predation in 3500 ml beakers increased as anchovy larvae 
concentration was raised. Two of the copepods, Labidocera jollae and L . 
trispinosa , were only efficient as predators on yolk-sac larvae, but a third 
species, Pontellopsis occidentalism also was able to prey on more developed, 
faster swimming larvae. The presence of alternate prey (Artemia salina nauplii) 
reduced larval mortality caused by Labidocera spp. in direct proportion to the 
numbers of Artemia that were present. In similar experiments, but using the 
euphausiid Euphausia pacifica , as an anchovy larva predator, Theilacker and 
Lasker (94) demonstrated that larval, juvenile and adult stages of the 
euphausiid could meet their daily carbon requirements by preying upon 
northern anchovy yolk-sac larvae. There was no strong evidence that anchovy 
concentration, when above 10 per 3500 ml, or the presence of alternate prey 
(Artemia nauplii) significantly influenced the predation rate on anchovies, at 


187 


least by juvenile euphausiids. Euphausiids, like pontellid copepods, were most 
successful at capturing yolk-sac larvae. Based on laboratory results and known 
abundances of pontellid copepods, euphausiids, and yolk-sac anchovy larvae in 
surface waters off the coast of California, it is possible that copepods and 
euphausiids can have a significant influence on anchovy larvae survival (62, 94). 

Yolk-sac larvae of the Pacific herring Clupea harengus pallasi were offered as 
prey to the amphipod Hyperoche medusanim in experiments conducted in 500 
ml beakers (99). The numbers of larvae preyed upon increased as both predator 
and larval densities increased. But, the number of prey attacked per hour per 
predator decreased as predator abundance increased. The number of herring 
larvae attacked per hour increased, but the rate of increased slowed as the 
concentration of herring larvae was raised. When “flatfish” larvae were 
provided as alternate prey, the amphipods showed a preference for herring. 
Amphipods such as Hyperoche medusanim may be an important source of 
mortality to Pacific herring larvae in the sea, especially when the 
newly-hatched larvae are concentrated near the spawning areas. 

Kuhlmann (54) investigated the chaetognaths Sagitta setosa and S. elegans 
and their possible role as predators on several species of fish larvae. Despite the 
often observed phenomenon in plankton samples of larval fish in chaetognath 
guts, S. setosa and S. elegans did not prefer the fish larvae in laboratory 
experiments when copepod prey was present in sufficient quantity. Kuhlmann 
(54) did not believe that the chaetognaths were important predators on larval 
fishes. He did find that both S. setosa and S. elegans consistently ate fish larvae 
after starvation periods of 24 to 48 hours if copepods were not offered as 
alternate prey. 

ROLE OF NATURAL PHYSICAL AND CHEMICAL VARIABLES 

A review of literature dealing with effects of natural environmental factors 
on development of marine fishes reveals a wealth of information on egg and 
yolk-sac stages (e.g., 14, 36, 38). However, these factors have not been 
intensively studied for larval stages from the time of first feeding to transition 
to the juvenile. 

Light 

Blaxter (16, 18) discussed the preferences of fish for light of specific 
intensities. This preferendum may vary from day to night and may not be 
available at the preferred intensity in some shallow water situations where little 
vertical migration is possible. The evidence reviewed by Blaxter (16) 
demonstrates that the light preferendum is variable among species and also 
among individuals. 


188 


Marine fish larvae typically are visual feeders and require a minimal light 
intensity above 10 "-10 lux to feed optimally (14, 16). Light levels reported 
by aquaculturists for successful culture of marine fish larvae have ranged from 
250-10,000 lux (9, 42). A 500-3000 lux range has been used most often. A 


minimum light intensity is necessary for initial detection of prey, visual 
recognition and prey selection. At light intensities close to the threshold level 
there is a gradual reduction in larval activity and in feeding performance (16). 
Blaxter (19) recently has summarized information on the anatomy of eyes in 
fish larvae and also has discussed the development of vision. A pure cone 
retina, which requires relatively high light intensities to be effective, appears to 
be typical of larval stages of fish and such retinas have been identified in the 
Atlantic herring (22), the plaice (12) and other species (19, 23). In general, for 
the few species of larvae that have been studied, the light intensity range in 
which feeding activity decreases is approximately 10 1 -10" 1 lux (11, 12, 13). 
This is near the dusk-dawn light intensity range of 10"-10’^ lux (16). 


Laurence (60) estimated the number of hours required daily by winter 
flounder larvae at 8°C to consume a ration that exceeds the maintenance 
ration. For a prey concentration of 3.4 cal/1 (= 500 copepod nauplii/1), the 
minimum suitable for survival and growth, first feeding winter flounder larvae 
would require about 20 hours per day to consume the maintenance ration. The 
diurnal light period, when light intensities are above 10" lux, is most important 
at low prey concentrations. For prey concentrations exceeding 6.8 cal/1 (= 
1000 nauplii/1) winter flounder larvae could meet their daily food 
requirements in less than 10 hours and at prey levels above 13.6 cal/1 (= 2000 
nauplii/1) established feeders could meet requirements in only 5 hours. It is 
apparent that the seasonal variation in day length and light intensity are 
important elements in any model predicting survival of fish larvae, particularly 
in high latitudes where seasonal variation is greatest. 


Possible harmful effects of natural sunlight on larvae are poorly known. 
Ultraviolet light near the sea surface may be deleterious to pelagic eggs (63). 
Some information on effects of ultraviolet light on pelagic embryos may apply 
to larval stages. Pommeranz (75) tested effects of ultraviolet light on plaice 
embryos, using an artificial UV intensity of 0.05 ly/min (1 ly n 1 cal/cm") at 
the water surface of 350 ml incubators with a 200 ml/min water exchange. 
Although results were not conclusive, lower percentages of embryos survived in 
12 hours and 24 hour-exposed incubators than in control incubators which 
were in the dark. Pommeranz (75) also exposed plaice embryos to natural 
daylight, natural daylight with UV wavelengths filtered out, and natural 
daylight with long wave infrared above 1400 nm filtered out. Average light 
intensities in two experiments were 257 ly/day and 462 ly/day. Only the high 
intensity experiment caused high mortality of embryos (35 percent). 
Ultraviolet light was considered to be the lethal agent because corresponding 
mortalities were not observed in incubators where ultraviolet was filtered out. 


189 


Temperature 

Brett (25) refers to temperature as a polymorphic environmental factor that 
may be a lethal agent, a controlling factor regulating metabolism and 
development, a limiting factor restricting activity and distribution, a masking 
factor interacting with other environmental factors, or a directing agent such as 
a thermal gradient. It appears that the major temperature effect on marine fish 
larvae is that of a controlling factor regulating metabolic and developmental 
rates. In turn, those rates can affect survival of larvae through their influence 
on establishment of exogenous feeding and regulation of food requirements 
(e.g. 43, 57, 59, 60). For clupeiform, perciform, and pleuronectiform larvae, a 
6-10°C range has been reported in which culture attempts are most successful 
(8, 10, 43, 57, 59, 61), although some survival can be obtained over wider 
temperature ranges. 

There are few temperature effect-metabolic rate studies on marine fish 
larvae. Laurence (59) examined growth and metabolism of feeding winter 
flounder larvae at 2°, 5° and 8°C. Larvae reared at 5° and 8°C were tested 
until metamorphosis and the specific growth rate at 8°C (10.1 percent/day) 
was significantly higher than that at 5°C (5.8 percent/day). The growth rate at 
2°C (2.6 percent/day) was less than at 5°C but not significantly less. 
Metamorphosis took 49 days and 80 days at 8° and 5°C, respectively. At 2°C 
larvae did not survive more than six weeks after yolk absorption. Power 
functions describing oxygen consumption of winter flounder in relation to 
body weight had exponential coefficients lower than the expected theoretical 
value of 0.80 (0.49 for 8°C, 0.56 for 5°C, 0.54 for 2°C). When separate power 
functions were fitted for larvae and for metamorphosed juveniles, the 
exponential coefficients for larvae closely agreed with the theoretical 0.80 
value for all three temperatures, but the coefficients for metamorphosed 
juveniles were lower. 

Hoss et al (41) examined the effect of a rapid 12°C rise in temperature 
(thermal shock) on growth of pinfish Lagodon rhomboides and spot 
Leiostomus xanthums, and oxygen consumption of pinfish, to determine if 
growth and metabolism could be used to detect sublethal effects of power 
plant thermal pollution. The fish that they used were transformed juveniles, in 
most respects (5.15-7.89 mg dry weight for pinfish, 11.23-23.70 mg for spot). 
No significant difference in growth was observed for thermally shocked and 
control groups. Oxygen consumption rates of experimental and control pinfish 
indicated that a 12°C shock produced a slight increase in consumption rate 
which returned to normal levels within a few hours. Their determinations (41) 
of critical thermal maxima and survival after acute thermal shocks may not 
represent responses that might be obtained for smaller larvae. 
Time-temperature exposure histories are critical for determining thermal 


190 


effects of entrainment (87, 89), but there are no such studies that include 
marine fish larvae from first feeding to metamorphosis stages. 

During the past 15 years temperature responses often have been investigated 
in conjunction with effects of other environmental factors, usually variations in 
salinity for embryos and yolk-sac larvae (e.g. 66, 86). Multi-dimensional 
analysis has led to use of response surface models which permit evaluation of 
interacting effects such as between temperature, salinity, oxygen, and dose 
time (1). However, most of this research has dealt with the egg, embryo, and 
pre-feeding larval stages (e.g. 2, 3, 4, 5, 68). 

Salinity 

The developing eggs and yolk-sac larvae of many marine teleosts are known 
to tolerate wider ranges of salinity than they are likely to encounter under 
natural conditions (e.g. 2, 5, 36, 38, 68, 77, 86), but there are few studies 
dealing with salinity tolerances of typical pelagic marine fish larvae during the 
actively feeding stages. 

In unaltered environments, the effect of changes in salinity on larval survival 
may be minimal, since pelagic larvae usually will be retained within a water 
mass that does not undergo extreme salinity changes. In the lower latitudes, 
where time for larval development to metamorphosis is short, the probability 
of an extreme salinity change that might cause mortality seems even less 
probable than in higher latitudes. Holliday (36), in reviewing data on salinity 
tolerances of Atlantic herring and plaice, observed that newly hatched larvae 
had a wider tolerance range for salinity than did metamorphosed juveniles. 
Tolerance to high salinities decreased from about 60°/oo at hatching to about 
40°/oo after metamorphosis, while low salinity tolerance changed little during 
development, ranging from about 2-8°/oo for both species. Kurata (55) 
obtained similar results for Pacific herring, C. harengus pallasi larvae which 
could tolerate a salinity range of approximately 2-60°/oo at 10 days after 
hatching, but only 2-42°/oo at 20 days. 

There are several investigations on salinity tolerances of non-typical or 
non-pelagic marine fish larvae, from which conclusions about tolerances of 
marine fish larvae in general perhaps can be inferred. For mummichogs 
Fundulus heteroclitus the range of salinity tolerance was very wide, 
0.39-100.00°/oo (51). California killifish larvae F. parvipinnis also had a wide 
salinity tolerance, but the tolerance for low salinities decreased with age (76). 
Two atherinids, the California grunion Leuresthes tenuis and the Gulf grunion 
L. sardina , were tested for salinity tolerances during the larval stage (78, 79). 
Gulf grunion had a wider salinity tolerance range than did California grunion, 
but in both species the tolerance range decreased with age. A reasonable 


191 


conclusion, based on limited data, is that newly hatched larvae of marine fishes 
are unlikely to suffer mortality as a direct effect of salintiy, but that older 
larvae are more vulnerable and could be killed by physiological stresses caused 
by salinity extremes. 

Oxygen uptake of anesthetized Atlantic herring eggs and newly hatched 
larvae did not differ significantly at test salinities of 5, 15, 35 and 50 /oo (38). 
For larvae there was variable oxygen uptake, the rates sometimes being 10X 
the pre-transfer oxygen consumption rate. For example, for a transfer from 35 
to 5°/oo at 8°C, larval oxygen consumption went from about 0.07 M1 
0-,/larva/hour to as high as 0.7 jul 0->/larva/hour within one hour after 
transfer. Oxygen uptake then fluctuated before returning to normal about five 
hours after transfer to the test salinity. Such fluctuations occurred for six-eight 
hours following transfer and were believed caused by osmoregulatory 
imbalance prior to acclimation to the treatment salinity. 

Dissolved Oxygen 

Vernberg (97) remarked that effects of low oxygen levels on animals are not 
easily determined under natural conditions because anoxic situations are 
always accompanied by other factors such as increased carbon dioxide and 
hydrogen sulfide concentrations. The effects of temperature and salinity on the 
solubility of oxygen also complicate the analysis of direct oxygen effect. 

Dissolved oxygen requirements of developing eggs and larvae of Salmonidae 
and other freshwater or estuarine species have been investigated many times 
(e.g. 35, 91, 98). There are few studies on marine fish larvae to determine their 
tolerances to low oxygen tensions (30, 85). De Silva and Tytler (30) found that 
the incipient lethal oxygen level (LD^q) for Atlantic herring and plaice varied 
with development from the yolk-sac stage to metamorphosis. At 10°C the 
LD^q for yolk-sac larvae was 1.93 ml/1 for herring and 2.73 ml/1 for plaice. 
After larvae had been feeding for two weeks the LD^q was 3.08 and 2.66 ml/1 
respectively. At 56-63 days after hatching for herring and 4249 days after 
hatching for plaice, gills developed and the LD^q levels fell to 2.91 and 2.52 
ml/1, respectively. At metamorphosis, 70-80 days after hatching for herring 
and 77-84 days after hatching for plaice, the LD^q was 2.17 and 1.69 ml/1, 
respectively. De Silva and Tytler (30) also measured routine metabolism of 
herring larvae from 7-62 days after hatching and plaice larvae from 5-75 days 
after hatching at 10°C. For the relationship between oxygen consumption and 
weight, they obtained exponential coefficients of 0.82 for herring and 0.65 for 
plaice. These values are higher than the values 0.49-0.56 obtained by Laurence 
(59) for winter flounder from hatching through metamorphosis, although 
Laurence obtained a coefficient of 0.80 when he excluded metamorphosed 
individuals from his analysis. 


192 


In surface waters of the euphotic zone dissolved oxygen usually ranges from 
4-8 ml/1 with supersaturation (> 6-9 ml/1) possible in highly productive 
shallow coastal waters. Kalle (52) reported that in coastal areas with high 
primary production, oxygen super-saturations up to 120 percent are not 
unusual during periods of intensive solar illumination. In shallow waters, 
temporary super-saturations may approach 500 percent (52). Mortality or 
stress of fish larvae due to low oxygen tensions probably occurs only under 
unusual conditions in the sea. 

Miscellaneous Environmental Factors 

Environmental factors such as turbidity, mechanical stresses, and shear 
forces likely to be found in nature have not been studied experimentally with 
regard to effects on marine fish larvae. A few investigations of effects of these 
factors on embryonic stages indicate that embryos are resistant to high 
sediment suspensions and mechanical forces which are present in the 
environment (e.g. 71, 75, 88). 

Schubel et al (88) observed that striped bass Morone saxatilis eggs could 
tolerate silt loads up to 500 mg/1. They noted that turbidity in areas being 
dredged could be as high as 1000 mg/1, which would cause significant embryo 
mortality, but that such high concentrations rarely occurred. Hoss et al (40) 
tested larvae of seven estuarine species with three concentrations of sediment 
extracts (the supernatent from 500 g of Charleston Harbor sediment shaken in 
one liter of filtered seawater). Under their laboratory conditions, survival of 
larval pinfish and menhaden Brevoortia tyrannus was 25-0 percent at the 75 
and 100 percent test concentrations. The supernatent water and sediments 
were not analyzed for toxic substances by the authors, but they cited 
references to relatively high levels of lead, copper, zinc and chromium in 
Charleston Harbor sediments. 

Pommeranz (75) investigated mechanical properties of plaice eggs by 
deforming them with a lever. The force to burst the chorion varied with time 
from fertilization and ranged from about 1.5 g during the 30 minutes after 
fertilization to a mean of about 700 g and 600 g for gastrula and embryo 
stages, respectively. For comparison purposes, Pommeranz (75) cited one 
rough estimate of the pressures developed by a spilling breaker in the open sea 
as approximately 0.1 kg/cm". 

Morgan et al (71) subjected striped bass and white perch Morone americana 
embryos and yolk-sac larvae to experimental shear forces of 0-86 dynes/cm 2 
over exposure times of 1-20 minutes. The estimated median lethal shear (LS^q) 
that could kill 50 percent of the embryos and larvae^ ranged from 120 
dynes/cm 2 for a 20 minute exposure, to 785 dynes/cm 2 for a one minute 


193 


exposure. Estimated LS^q values for striped bass yold-sac larvae were 785 
dynes/cm 2 and 300 dynes/cm 2 for 1 and 4 minute exposures, respectively. 
White perch yolk-sac larvae were more vulnerable, their LS^g values were 415 
dynes/cm 2 and 125 dynes/cm 2 for 1 and 4 minute exposures respectively. 
Calculated average shear force in the Chesapeake and Delaware Canal, where 
striped bass eggs occur, was only 13.8 dynes/cm~, far below the estimated 
LS 50 values. The authors (17) also related these LS 5Q values to expected shear 
forces of 72-230 dynes/cm 2 that might be present in the water box of a power 
plant cooling system. The 230 dynes/cm 2 shear approaches the 4 minute LS 5 q 
value for striped bass yolk-sac larvae and exceeds it for white perch. 

Chipman (27) reviewed literature on effects of naturally occurring ionizing 
radiation on marine animals. He found no convincing evidence to demonstrate 
that marine animals showed any response, functional or structural, to ionizing 
radiation levels present in the environment. In marine animals observable 
effects are primarily at the cellular level, and the radiation tolerance is a 
function of the dose-rate, time patterns of exposure and metabolic rate; 
consequently, effects would be most evident during embryonic development 
(27). 

FUTURE RESEARCH 

Both laboratory and transitional laboratory-field studies will extend our 
knowledge of environmental effects on larval stages of marine fish. A recent 
colloquium on larval mortality and the recruitment problem has defined some 
areas in need of research (48). Emphasis of that colloquium was to advocate 
research related to starvation and predation, the two factors that probably have 
the greatest effect on recruitment of year classes. Environmental stresses from 
man’s activities are additional threats, particularly to estuarine species or those 
found over the continental shelf. Pollution effects on embryos can cause gross 
functional and structural abnormalities that may produce yolk-sac larvae 
incapable of surviving to the exogenous feeding stage (81). Larvae can be 
equally vulnerable to deleterious effects of pollutants, and their responses to 
this stress may be reflected in impaired predator avoidance behavior and food 
capture efficiency. More subtle effects could involve functional disruptions of 
metabolism, temperature and salinity tolerance, and enzyme-substrate 
interactions. Both direct and indirect effects of environmental modification on 
recruitment need to be determined. 

The ability to culture larvae widens the possibilities for laboratory research 
which will help interpret results of field studies. The larval stage is a dynamic 
one, characterized by fast growth, sometimes spectacular developmental 
changes, and frequent shifts in behavior. Typical toxicity bioassays, where 
times to 50 percent mortality are estimated, may not be the best approach to 


194 


determine how environmental factors affect survival of a larval cohort. 
Environmental factors act in concert, and it is the sum of experiences over the 
entire embryo and larval stages that determines whether a good or poor year 
class results. A bioassay for 96 hours, testing one or two factors, usually can 
provide only a rough evaluation of the potential effect of the factor(s) on 
recruitment. More meaningful conclusions can be drawn from investigations 
that encompass the entire larval period. Many studies of that kind have been 
carried out on larvae of freshwater fishes (69), but the difficulties in rearing 
larvae of marine species have limited most bioassay research to embryo and 
yolk-sac larva stages. 

Experiments in large volumes of seawater, either in plastic bag enclosures, 
such as those used in recent Controlled Ecosystems Pollution Experiments 
(CEPEX) (70) or in large tanks (74) hold great promise because whole 
communities can be entrapped in such volumes. Effects of predation and 
competition can be evaluated. Direct and indirect effects of added pollutants 
on each trophic level can be observed. Recruitment success or failure by fishes 
in such systems can be interpreted in the context of observed changes that 
took place in the plankton community during the course of larval 
development. 

Other approaches include transitional studies that combine laboratory and 
field experiments. The “field bioassay” developed by Lasker (56) uses 
laboratory-reared larvae in shipboard experiments, in which larvae are reared in 
natural seawater sources to evaluate the potential of particular water masses to 
support larval survival and growth. The recent discovery, based on laboratory 
studies, that daily growth rings are present on otoliths of larvae, will allow 
better estimates of larval growth and mortality rates in the sea (26, 93), and 
also will allow comparison of growth in the laboratory with growth under 
natural conditions. 

Except for swimming-feeding behavior of a few species and behavioral 
responses to varying light levels (12, 15, 17) little is known about normal 
behavior patterns of larvae or changes in behavior induced by environmental 
effects. Behavioral studies not only can increase our understanding of how 
pollutants affect larval behavior, but they also can provide important insight 
into how predation and competition operate during the larval stage. 

There are many techniques presently available that allow environmental 
factors and their effects on marine fish larvae to be evaluated. In the next 10 
years, culture of marine fishes will be routine procedure at many laboratories; 
and as more data accumulate, some of the seemingly contradictory results 
obtained to date, especially with regard to critical food concentrations, will be 
resolved. Additional species of marine fishes need to be tested for larval 


195 


tolerances to environmental factors. Present day literature is dominated by 
research on herring, plaice, and northern anchovy; the first two species are 
rather atypical pelagic, marine fish larvae because of their unusually large size 
and advanced development at hatching. Refinement of culture methods, 
improved techniques for handling and testing delicate larvae, and examination 
of multiple factor effects and interactions throughout the period of larval 
development will help us to better understand how the environment acts on a 
cohort of larvae. This knowledge can be incorporated into predictions of 
recruitment success based on probable influences of environmental factors 
operating during the larval stage. 

ACKNOWLEDGEMENTS 

Support from Environmental Protection Agency Grant R804519 made the 
preparation of this paper possible. 


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(Ayres): a Comparison with Gulf Grunion, L. sardina (Jenkins and 
Evermann). J. Exp. Mar. Biol. Ecol. 24: 73-82. 

80. Riley, J.D. 1966. Marine Fish Culture in Britain. VII. Plaice ( Pleuronectes 
platessa L.) Post-Larval Feeding on Artemia salina L. Nauplii and the 
Effects of Varying Feeding Levels. J. Cons. Int. Explor. Mer 30: 204-221. 

81. Rosenthal, H. and D.F. Alderdice. 1976. Sublethal Effects of 
Environmental Stressors, Natural and Pollutional, on Marine Fish Eggs 
and Larvae. J. Fish. Res. Board Can. 33: 2047-2065. 

82. Rosenthal, H. and G. Hempel. 1970. Experimental Studies in Feeding and 
Food Requirements of Herring Larvae ( Clupea harengus L.), pp. 344-364. 
In: J.H. Steele (ed.) Marine Food Chains. Univ. Calif. Press, Berkeley. 

83. Rosenthal, H. and G. Hempel. 1971. Experimental Estimates of Minimum 
Food Density for Herring Larvae. Cons. Int. Explor. Mer, Rapp, et Process 
Verb. 160: 125-127. 

84. Saksena, V.P. and E.D. Houde. 1972. Effect of Food Level on the Growth 
and Survival of Laboratory-Reared Larvae of Bay Anchovy ( Anchoa 
mitchilli Valenciennes) and Scaled Sardine ( Harengula pensacolae Goode 
and Bean). J. Exp. Mar. Biol. Ecol. 8: 249-258. 

85. Saksena, V.P. and E.B. Joseph. 1972. Dissolved Oxygen Requirements of 
Newly Hatched Larvae of the Striped Blenny (Chasmodes bosquianus), 
the Naked Goby ( Gobiosoma bosci), and the Skilletflsh ( Gobiesox 
strumosus). Ches. Sci. 13: 23-28. 


203 


86. Santerre, M.T. 1976. Effects of Temperature and Salinity on the Eggs and 
Early Larvae of Caranx mate (Cuv. & Valenc.) (Pisces: Carangidae) in 
Hawaii. J. Exp. Mar. Biol. Ecol. 21: 51-68. 

87. Schubel, J.R. 1975. Some Comments on the Thermal Effects of Power 
Plants on Fish Eggs and Larvae, pp. 31-54. In: S.B. Saila (ed.) Fisheries 
and Energy Production: A Symposium. Lexington Books, Lexington, 
Mass. 

88. Schubel, J.R., A.H. Auld, and G.M. Schmidt. 1974. Effects of Suspended 
Sediment on the Development and Hatching Success of Yellow Perch and 
Striped Bass Eggs. The Johns Hopkins University, Chesapeake Bay 
Institute Spec. Rep. 35, Ref. 74-2, 12 p. 

89. Schubel, J.R., C.F. Smith and T.S.Y. Koo. 1977. Thermal Effects of 
Power Plant Entrainment on Survival of Larval Fishes: A Laboratory 
Assessment. Chesapeake Sci. 18: 290-298. 

90. Shirota, A. 1970. Studies on the Mouth Size of Fish Larvae. Bull. Jap. 
Soc. Sci. Fish. 36: 353-368. 

91.Siefert, R.E., W.A. Spoor, and R.F. Syrett. 1973. Effects of Reduced 
Oxygen Concentration on Northern Pike (Esox lucius ) Embryos and 
Larvae. J. Fish. Res. Board Can. 30: 849-852. 

92. Stepien, W.P., Jr. 1976. Feeding of Laboratory-Reared Larvae of the Sea 
Bream Archosargus rhomboidalis (Sparidae). Mar. Biol. 38: 1-16. 

93. Struhsaker, P. and J.H. Uchiyama. 1976. Age and Growth of the Nehu, 
Stolephorus purpureus (Pisces: Engraulidae), from the Hawaiian Islands as 
Indicated by Daily Growth Increments of Sagittae. Fish. Bull., U.S. 74: 
9-17. 


94. Theilacker, G.H. and R. Lasker. 1974. Laboratory Studies of Predation by 
Euphausiid shrimps on Fish Larvae, pp. 287-299. In: J.H.S. Blaxter (ed.) 
The Early Life History of Fish. Springer-Verlag, New York, Heidelberg, 
Berlin. 

95. Theilacker, G.H. and M.F. McMaster. 1971. Mass Culture of the Rotifer 
Brachionus plicatilis and its Evaluation as a Food for Larval Anchovies. 
Mar. Biol. 10: 183-188. 


204 


96. Umeda, S. and A. Ochiai. 1975. On the Histological Structure and 
Function of Digestive Organs of the Fed and Starved Larvae of the 
Yellowtail, Seriola quinqueradiata. Jap. J. Ichthyol. 21: 213-219. 

97. Vernberg, F.J. 1972. Dissolved Gases, 9.3 Animals, pp. 1491-1526. In: 0. 
Kinne (ed.) Marine Ecology, Vol. 1, Pt. 3. Wiley Interscience, New York. 

98. Voyer, R.A. and R.J. Hennekey. 1972. Effects of Dissolved Oxygen on 
Two Life Stages of the Mummichog. Prog. Fish-Cult. 34: 222-225. 

99. Westernhagen, H. and H. Rosenthal. 1976. Predator-Prey Relationship 
between Pacific Herring, Clupea harengus pallasi, Larvae and a Predatory 
Hyperiid Amphipod, Hyperoche medusanun. Fish. Bull., U.S. 74: 
669-674. 

100. Wyatt, T. 1972. Some Effects of Food Density on the Growth and 
Behaviour of Plaice Larvae. Mar. Biol. 14: 210-216. 


205 


LABORATORY CULTURE OF THE 
GRASS SHRIMP 
Palaemonetes vulgaris 

by 

Thomas E. Bigford 
Environmental Research Laboratory 
United States Environmental Protection Agency 
Narragansett, Rhode Island 02882 


ABSTRACT 

Experiments have been undertaken to test the feasibility of hatching, 
rearing, and breeding an in-laboratory population of the grass shrimp, 
Palaemonetes vulgaris. Primary objectives include continual availability of all 
life stages (for use in experiments or as food organisms) and comparisons of 
lab-reared and field-collected animals. 

Systems have been designed for culturing the grass shrimp throughout its 
life cycle. Larval survival percentages reached 70 percent in the beaker and 
“hatching jar” culture systems. Up to 75 percent of these metamorphosing 
larvae survived to adult stages. Both Artemia salina and the flake food Tetra 
Marin were proven to be successful diets for P. vulgaris . 

Results indicate that P. vulgaris can be maintained and propagated in the 
laboratory. Larvae hatched in the lab have been induced to produce normal 
larvae within as little as 90 days. This generation time is apparently shorter 
than the time in field populations. 

INTRODUCTION 

Most marine biology research efforts require a consistent supply of 
experimental animals. Field-collected organisms often confer variability due to 
individual differences in life history, nutrition, etc. Many of these problems can 
be controlled by culturing the animals under rigorous, well-documented 
laboratory conditions. 

The purpose of this study was to develop and standardize laboratory 
holding and culture techniques for the grass shrimp, Palaemonetes vulgaris 
(Say). Establishment of suitable methods would permit testing of the 


206 


feasibility of hatching, rearing and breeding a laboratory population of the 
shrimp for use as experimental animals. Of primary consideration in this study 
was the development of flow-through culture systems for the various life 
stages. Static designs represent poor simulations of field conditions and may 
impose unnecessary stresses on the animals (8). A secondary concern was to 
determine suitable diets for the juvenile and adult grass shrimp; Broad (1) 
found brine shrimp, Artemia salina , nauplii to be a very satisfactory larval 
food. Success for the project must be measured in terms of growth, survival, 
and population reproduction. 

The advantages of using lab-reared organisms are countered by several 
anomalous characteristics of organisms maintained in the lab. Morphological 
changes, as compared to field animals, have been noted by Paul Yevich 
(Environmental Research Lab, Narragansett, R.I.; personal communication) in 
many marine animals. However, the increased control of age, nutrition, and 
prior exposure to environmental variables would appear to outweigh slight 
changes in morphology and behavior. 

The grass shrimp, Palaemonetes vulgaris (Say), was selected for these studies 
for several reasons: the shrimp is a common estaurine species available to 
researchers along the Atlantic and Gulf of Mexico coasts (9); the animal is 
relatively easy to rear in the laboratory; and the life cycle can be greatly 
compressed in the lab (4). 

EXPERIMENTAL 

Several ovigerous grass shrimp were collected by dip net on 14 July 1976 in 
the Pettaquamscut River estuary adjacent to Narragansett Bay, Rhode Island. 
Mid-summer salinities at the collection site range from 25-30o/oo depending 
on the tidal cycle; water temperature was 21.5°C. 

Egg-bearing females were isolated in six C (1.6 U.S. gal.) tubs^ at 21.0- 
23.5°C and oceanic salinities (29 T4.5o/oo). Photoperiod was maintained at 
ambient levels of LI4:DIO. Water was changed daily and aerated gently. 
Shrimp were offered food during this holding period but rarely fed. 

Larvae hatched after 1 to 17 days of holding and were immediately pipetted 
into the flow-through system shown in Figure 14-1. About 200 larvae were 
held in this two £ system for 21 days, by which time all shrimp had reached the 
late larval stages. Developing larvae were fed excess quantities of newly hatched 
and one-day old brine shrimp nauplii, Artemia salina (San Francisco Bay 
Brand). 


1 Rubbermaid Commercial Products, Inc., Winchester, VA. 


207 



h 2 o 


AIR INPUT INFLOW FOOD INPUT 



h 2 o OUTFLOW 


AIRSTONE 

WATER 

CURRENTS 


PETRIE DISH COVER 


SPRING 


WATER LEVEL 


GLASS OUTER SLEEVE 


GLASS INNER SLEEVE 



NYLON MESH 


SILASTIC TUBING 

CLAMP 


Figure 14-1. The 2 8 Flow-Through Beaker System. 

NOTE: Designed by Dr. W. B. Vernberg under E.P.A. Grant R 802071 and 
modified for use in the culture of larval and juvenile Palaemonetes vulgaris. 


All surviving juvenile P. vulgaris (about 140) were transferred on day 22 
(post hatch) to the 20 8 “hatching jar”“ commonly used in hatching and 
rearing larval fish (Figure 14-2). This 30 cm diameter, clear acrylic tank, was 
modified from the manufacturer’s design by placing a 400 jum nylon mesh 
across the outflow ramp, thereby eliminating the need for a mesh over the top 
of the entire system. This smaller outflow area effectively decreased the 
chances of impinging larvae on the mesh. Gentle aeration and a water flow of 
50 ml/min created a satisfactory circulation pattern. This hatching jar system 


“Midland Plastics Co., Brookfield, Wl. 


208 































AIR 

INFLOW 


SEAWATER 

INFLOW 



Figure 14-2. The 12 C Hatching Jar System Used to Culture 
Juvenile and Adult Grass Shrimp, Palaemonetes vulgaris. 


NOTE: 6 and 48 £ sizes are identical in design. 


was used for 14 weeks, during which time a combination of thawed and live 
juvenile brine shrimp were fed in excess, with unconsumed food removed by 
siphon once each week. 


All grass shrimp were transferred from the 20 £ jar at age 17 weeks. Of these 
shrimp, 40 were used in a diet study, and the remaining 66 were placed in a 48 
£ hatching jar scaled-up version of the 20 £ size (Figure 14-2). In the diet study, 
40 shrimp were divided evenly between two 4 2 systems resembling that shown 
in Figure 14-1. One group of 20 shrimp was reared on lab-reared Artemia 
adults while the other group was fed 243 400 jum pieces of the commercial 
flake Fish food Tetra Marin. Animals were fed daily in excess. Growth in 
carapace length, survival, and the incidence of ovigerous females were 
considered in determining the suitability of each diet. 


209 


























RESULTS AND DISCUSSION 


Attempts to culture and maintain a laboratory population of P. vulgaris 
have been successful thus far. Ovigerous females were obtained from adults 
hatched in the laboratory and cultured for 90 days. These egg-bearing females 
have yielded morphologically normal larvae, thereby indicating that the eggs 
resulting from lab-reared females are viable. After 16 months of culture, a total 
of 21 ovigerous females have been collected from the system. Six of these 
shrimp are females that also bore eggs in the first laboratory spawning season 
(November, 1976 to January, 1977 or 9 to 10 months ago). Problems in 
controlling photoperiod and water temperature have limited successful hatches 
to only two females. Little (4) has discussed how manipulation of these two 
environmental factors can be used to induce winter breeding in grass shrimp. 

The diet study (Table 14-1) has indicated on a gross scale that a flake food 
can be used as a diet. Ovigerous females were collected from the four £ 
flow-through systems used for both the brine shrimp and Tetra Marin diets. 
Therefore, growth, survival and reproduction are acheivable with the live and 
dried foods. 

All of the systems and techniques mentioned herein have yielded 
satisfactory results. However, some minor problems remain. One such problem 
is cannibalism, especially in the 20 and 48 £ hatching jars used as holding tanks 
for juvenile and adult grass shrimp. Obvious solutions include increasing the 
food available, either as more food per day, or as multiple daily feedings, or 
decreasing the density of shrimp. A certain degree of cannibalism is to be 
expected in mass cultures during periods of molting. 

A second problem, also in the hatching jar systems, relates to the physical 
design of the container. The concave bottom of the jar, coupled with a circular 
flow, causes a centrifuging of the shrimp into the center near the bottom. A 
flatter bottom with a larger bottom surface area to volume ratio could be a 
solution. The 40 £ kriesel systems (3) used in lobster culture efforts have the 
desired flatter bottoms, and also jetted water inflow along the sides that create 
a more uniform distribution of the shrimp. Preliminary studies studies indicate 
that the kriesel design will be very successful for juveniles and adults. 
Compared to growth in the four £ beaker systems (See Table 14-1), the kriesel 
has yielded significantly higher growth rates. Mean carapace length in the 
kriesel after nine weeks was 6.8 ± 0.69 mm (range 5.9 to 7.9 mm), a size not 
attained until an age of about 20 weeks in the beaker. 

One last problem is animals flipping out of the systems, especially the 48 £ 
jar. This appears to happen in conjunction with a molt, and the subsequent 
cannibalism pressure from other shrimp in the system. A simple solution to the 


210 


Table 14-1. Summary of growth, measured via carapace length, 
and survival of juvenile and adult Palaemonetes vulqaris 

i 4 a ■ ■■ _ ** 



CL 

E 


-C 

to 


O 

to 
■M 

c 

D 

O £ 
O CL 

_ 03 

.2 ro 


3 


£ O 


■ • 
UJ 
H 
O 


211 


were 20 per four Si beaker. Carapace lengths were measured from tip of rostrum to posterior edge 






escape problem is a cover over the system. However, such a design may change 
the cause of mortality from escape and desiccation to cannibalism. 

Each of these systems emphasizes low maintenance and unlimited scale-up 
potential. Care was taken during the design phase to avoid sharp corners, excess 
mesh area, or eddying currents. Hartman (2) was shown that brachyuran larvae 
become impinged in corners that break spines or setae and impede molting. He 
also mentioned the importance of tapered walls so that larvae and food do not 
become caught in eddying currents. 

Even with these problems, survival of all life stages has been high. Larvae 
cultured in the beaker systems have shown approximately 70 percent survival 
when fed Artemia nauplii in excess. In this experiment, survival of juveniles 
and adults reared in the 20 2 hatching jar was 75 percent (since 
metamorphosis) over a 14 week period. The diet studies have shown similar 
survivals in smaller cultures. 

Several other comments are worthy of mention. The ages at transfer from 
one system to another represent the schedule used in this study and most likely 
could be altered without problems. Also, one key factor to consider in the two 
2 and four 2 flow-through systems and the hatching jars, is mesh size. A mesh 
should be chosen that will permit debris to pass through, yet retain both larvae 
and food organisms. For these reasons a 243 or 400 jum mesh was used. 

Use of A. salina nauplii as a diet for larval grass shrimp has been 
substantiated by several investigations (1, 6). The study by Broad (1) 
confirmed that diets including brine shrimp were more successful in terms of 
survival and development than diets lacking this animal tissue. Provenzano and 
Goy (6) established the possibility of using Artemia from several locations, 
including San Francisco, Canada and, to a lesser degree, Utah. 

Establishment of a laboratory population of grass shrimp should lead to 
increased use of the animal in bioassays. Nimmo et al (5), based on cadmium 
bioassays, concluded that adult P. vulgaris were “acutely and chronically” 
more sensitive than the pink shrimp, Penaeus duorarum. Studies by Shealy and 
Sandifer (7) have shown the susceptibility of P. vulgaris larvae to mercury. 
Conversely, Vernberg et al (8) reported that P. pugio is quite resistant to 
cadmium bioassays. Apparently animal age and species are important; P. 
vulgaris may prove to be a better pollution indicator than P. pugio. 

REFERENCES 

1. Broad, A.C., 1957. The Relationship Between Diet and Larval Development 

of Palaemonetes. Biol. Bull. 112:162-170. 


212 


2. Hartman, M.C., 1977. A Mass Rearing System for the Culture of Brachyuran 
Crab Larvae. In: Proceedings of Eighth Annual Meeting of World 
Mariculture Society, Jan. 7-14, 1977. San Jose, Costa Rica. 

3. Hughes, J.T., R.A. Shleser and G. Tchobanoglous., 1974. A Rearing Tank 
for Lobster Larvae and Other Aquatic Species. Prog. Fish. Cult. 36:129-132. 

4. Little, G., 1968. Induced Winter Breeding and Larval Development in the 
Shrimp, Palaemonetes pugio Holthius (Caridea, Palaemonidae). Crustaceana 
2 (suppl.): 19-26. 

5. Nimmo, D.W.R., D.V. Lightner and L.H. Bahner, 1977. Effects of Cadmium 
on the Shrimps, Penaeus duorarum, Palaemonetes pugio, and Palaemonetes 
vulgaris. In (Ed. F.J. Vernberg et al) Physiological Responses of Marine 
Biota to Pollutants. Academic Press, New York. pg. 131-183. 

6. Provenzano, A.J., Jr. and J.W. Goy, 1976. Evaluation of a Sulphate Lake 
Strain of Artemia as a Food for Larvae of the Grass Shrimp, Palaemonetes 
pugio. Aquaculture 9:343-350. 

7. Shealy, M.H., Jr. and P.A. Sandifer, 1975. Effects of Mercury on Survival 
and Development of the Larval Grass Shrimp Palaemonetes vulgaris. Mar. 
Biol. 33:7-16. 

8. Vernberg, W.G., P.J. Decoursey, M. Kelley and D.M. Johns, 1977. Effects of 
Sublethal Concentrations of Cadmium on Adult Palaemonetes pugio under 
Static and Flow-Through Conditions. Bull. Environ. Contam. Toxical. 
17:16-24. 

9. Williams, A.B., 1965. Marine Decapod Crustaceans of the Carolinas. Fish. 
Bull. 65:1-298. 


213 


EVALUATION OF VARIOUS DIETS ON 
THE LIPID AND PROTEIN COMPOSITION 
OF EARLY LIFE STAGES OF THE 
ATLANTIC SILVERSIDE 


Kenneth L. Simpson, Leslie M. Richardson and 

Paul S. Schauer 

Department of Food Science and Technology, 
Nutrition and Dietetics 
University of Rhode Island 
Kingston, R.l. 02881 


ABSTRACT 

A study was performed to evaluate the effect of various natural and 
artificial diets on the lipid and protein composition of.laboratory cultured 
Atlantic silversides, Menidia menidia. Results were compared to analyses of 
wild silversides, which constituted the biochemical control. 

The best growth and survival of juvenile silversides was obtained on a live 
3-day-old brine shrimp nauplii diet. Substantially lower growth and survival 
were obtained on a freeze-dried brine shrimp diet and the artificial diets. 

Amino acids were incorporated into the tissue of batch cultured silversides 
fed a live 3-day-old brine shrimp diet by the fifth day of culture. Thereafter, 
the profiles changed very little, except for the levels of histidine and arginine in 
the* 58-day-old silversides. The amino acids of the cultured fish fed natural or 
artificial diets were quite similar. Bioavailability studies are necessary to 
ascertain the degree of incorporation and assimilation of dietary amino acids. 

The whole body fatty acid composition of cultured fish reflected the 
composition of their diets. Fish fed a live brine shrimp nauplii diet had higher 
total lipid levels and lower polyunsaturated fatty acid levels than wild 
silversides. Cultured fish may store large amounts of lipids in order to facilitate 
the bioaccumulation of long chain polyunsaturated fatty acids. The 
incorporation of cod liver oil into a diet previously containing a soybean oil 
increased the levels of the polyunsaturated fatty acids in the fish. The resulting 
fatty acid tissue levels resembled the long chain fatty acids of the wild fish 
lipids more closely than the profiles of fish fed brine shrimp nauplii. 


214 


INTRODUCTION 


The Atlantic silverside, Menidia menidia , is a marine fish used in bioassay 
studies due to its relative sensitivity to environmental contaminants (17). 
Bioassays using silversides have been relatively short-term studies, principally 
because of the dependence upon wild fish populations. Before long-term 
studies are possible, the dietary aspects of laboratory culture technology must 
be developed. The diet can affect the organism’s ability to respond in the a 
reproducible fashion. Additionally, the diet is an important feature in the 
ability of cultured fish to reach maturity, and spawn viable eggs necessary for 
multi-generation bioassay evaluations. 

Live brine shrimp, Artemia salina, have been used world-wide in the 
laboratory culture of larval marine fishes (4). Silversides used in toxicological 
bioassays by the Environmental Protection Agency’s Environmental Research 
Laboratories have commonly been fed brine shrimp as their primary diet. 
However, the difficulty of culturing large volumes of biochemically similar 
brine shrimp (8), coupled with increased costs and decreased availability (32) 
of cysts has mandated the need for an artificial diet. Providing an adequate, 
practical, and economical diet is a major factor limiting culture of most marine 
fishes reared on either a laboratory or commercial scale. Based on these facts, 
the University of Rhode Island, Food Science & Technology, Nutrition and 
Dietetics Department collaborated with the Environmental Research 
Laboratory to evaluate a number of artificial diets that could replace brine 
shrimp. 

In our study, we were attempting to produce a cultured fish that could 
respond in bioassays in a similar manner to wild fish, and provide comparable 
growth and survival as brine shrimp fed juvenile fish. This paper discusses the 
effects of various diets on the protein and lipid composition of laboratory 
reared silversides. 

EXPERIMENTAL 

General 

This study consisted of three parts: 1) a two month batch culture of 
silversides fed 3-day-old brine shrimp, 2) a preliminary evaluation of an 
artificial Atlantic Salmon, Salmo salar, diet comprised of a soybean oil base, 
and 3) an expanded study using brine shrimp and a number of artificial diets. 

Culture 

The collection of the gravid female silversides, the stripping and fertilization 
of the eggs, the hatching and feeding procedures, and the culture systems used 


215 


in this study have been previously documented (7). Gravid fish were collected 
from Bissel Cove, Narragansett Bay (R.I.) and trasported in aerated containers 
to holding tanks located at the Environmental Protection Agency Laboratory, 
Narragansett, R.I. Eggs were stripped from females onto nylon monofilament 
screens with a mesh size of 400jU, and fertilized by bathing them in the milt of 
two to three males (5). They were then suspended in egg hatching jars, 15 cm 
in diameter, modified from the original design of Buss (12) by the addition of a 
bottom center drain. 

After hatching, the fish were transferred to a 720 liter holding tank and ted 
live 3-day-old brine shrimp. Fish were periodically removed during the two 
month batch culture study for biochemical analyses. Fish used in both the 
preliminary and expanded diet evaluations were cultured for approximately 
two weeks. The jars used for hatching of the eggs, were also used as the culture 
vessels in the artificial diet studies. For the eight-diet expanded study each jar 
was stocked with 50, 23-day-old fish (individual mean weights, 8.90 mg) 
obtained from the batch culture population. Two replicates were run for each 
diet fed group. 

Diets and Feeding Procedures 

The wild plankton (Diet 1) were collected from a number of locations in the 
west passage of Narragansett Bay, R.I. and from local estuarine areas with a 
243ju mesh conical plankton net (Table 15-1). The plankton population was 
comprised of a mixture of copepods, primarily Acartia tonsa, and some 
invertebrate larvae (22). The plankton samples were transported to the 
laboratory in insulated containers and held at 20°C. 

The live brine shrimp nauplii (Diet 2) (San Francisco Bay Brand, USA) were 
incubated 12 to 24 hours in two liter separatory funnels containing filtered 
seawater (29.0 to 31.0 o/oo salinity, 20 to 22°C) and harvested after 72 hours. 
A starved group served as a control (Diet 3). The freeze-dried brine shrimp 
(Diet 4) was obtained by freezing the live brine shrimp to -38°C and then 
drying at 4jn/Hg pressure for 24 hours. 

Diets 5 through 9 were the artificial formulations. Tetra Marin (Diet 5) is a 
commercial flake diet used in aquarium fish applications and consists of 
unknown proportions of meals from fish, crab, mussel, lobster, beef heart, and 
brine shrimp. In addition, it is made up of such components as halibut liver, 
Calanus finmarchicus , kelp, oatflour, wheat germ, Agar-Agar , seaweed, and 
bone charcoal. The other four artificial diets were modified formulations 
originally prepared to suit the requirements of Atlantic salmon. The diets were 
prepared by the Tunison Laboratory of Fish Nutrition, U.S.F.W.S., Cortland, 
New York. The composition of these diets is given in Tables 15-2 and 15-3. 


216 


Table 15-1. Description of the Experimental Diets 


Experimental Diets 

Source and Description^ 

1. Wild plankton 

Collected in West Passage, Narragansett 

Bay by conical net with 243ju mesh opening 
and retained on a 116ju mesh sieve. 

2. Brine shrimp 
nauplii, live 

San Francisco Bay brine shrimp, Artemia 
salina, eggs hatched and harvested after 

72 hours in filtered and autoclaved seawater 
of 20-22°C and 20-30 o/oo salinity. 

3. Starved 

Unfed 

4. Brine shrimp 

nauplii, freeze-dried 

Nauplii as obtained in diet #2, then 
freeze-dried 24 hours to constant weight. 

5. Tetra Marin 

Lot #125244. Tetra Marin Staple Food, 

Tetra Werke Dr. rer. nat. Baensch, Melle, 

West Germany 

6. Artificial, CM-1 

Cortland #1 diet with cod liver oil. 

(See Table 1 5-2) 

7. Artificial, C-1 

Cortland #1 diet with soy bean oil. 

(See Table 1 5-2) 

8. Artificial, CMP-1 

Semi-purified diet with cod liver oil. 

(See Table 15-3) 

9. Artificial, CMP-2 

Semi-purified diet with cod liver oil and 
an amino acid supplement. (See Table 15-3) 


^ All diets were ground to a coarse powder of AOO/jl size or less. 

Fish were fed four times daily at a level of Five percent body wet weight. A 
compensation factor was provided to accommodate the flushing action of the 
system (7). 

Protein Analysis 

All fish were starved for 24 hours prior to sacrificing to reduce the stomach 
contents. The diet and Fish samples were acid hydrolyzed according to the 


217 





Table 15-2. Composition of the Artificial Diets, 

CM-1 and C-1 


Components 

Percent Composition 


CM-1 

C-1 

Herring meal 

40.00 

10.00 

Soy bean oil 

10.00 

10.00 

Corn gluten meal, 60% 

10.00 

10.00 

Wheat middlings, standard 

9.00 

9.00 

Brewers dried yeast 

5.00 

5.00 

Dried condensed fish solubles 

5.00 

5.00 

Dried Whey 

5.00 

5.00 

Meat and Bone Meal 

5.00 

5.00 

Soy bean oil 

— 

10.00 

Cod liver oil 

i 

Mineral mixture 

10.00 

— 

0.40 

0.40 

Vitamin mixture 

0.70 

0.70 


Mixture provided the following compounds in g/kg diet: MgSO^, 2.0; ZnSO^-l-^O, 0.3; 
FeS0 4 -7H 2 0, 0.3; CuS0 4# 0.3; KIO^ 0.0091 and MnSC> 4 -H 2 0, 1.0. 


Mixture provided 10,000 IU Vitamin A as retinyl palmitate; 4,000 IU Vitamin D as 
Cholecalciferol; 75 IU Vitamin E as dl-a-tocopheryl acetate; and the following amounts 
(milligrams) of other vitamins per kilogram of diet: menadione dimethylpyriminidol 
bisulfate, 10.0; thiamine HCL, 4.0; riboflavin, 30.0; calcium pantothenate, 150.0; 
niacinamide, 300.0; pyridoxine-HCL, 20.0; d-biotin, 6.0; folacin, 15.0; Vitamin B.^, 
0.002; L-ascorbic acid, 1000; inositol, 500.0; butylated hydroxytoluene (100%), 100.0; 
and choline chloride (70%), 1330.0. 


methods of Spackman et al (31) and Moore and Stein (27) with modifications 
by Niederwieser and Pataki (29), Blackburn (10) and Hirs (21). Amino acid 
analyses were performed on a Technicon Auto Analyzer (NC-2P) with a 25 cm 
column. An electronic integrator (Columbia Scientific Supergrator 2) was used 
to compute the absolute amounts of each amino acid. 

Protein content was assayed by microkjeldahl according to Hiller et al (20), 
and the moisture content was determined using procedures described by 
Chibnall et al (13). 


218 





Table 15-3. Composition of the Artificial Diets, 

CMP-1 and CMP-2 


Composition 

Percent Composition 


CMP-1 

CMP-2 

Casein 

40.00 

40.00 

Gelatin 

10.00 

10.00 

Cod liver oil 

10.00 

10.00 

Sucrose 

10.00 

10.00 

Dextrin, white Technical 

i 

Cellulose 

10.00 

10.00 

4.26 

7.16 

Choline chloride (70%) 

0.30 

0.30 

L-glutamic-HCI 

1.20 

— 

L-ascorbic acid 

0.30 

0.30 

L-glutamic acid 

6.30 

— 

NaCI 

0.50 

0.50 

Mineral mixture^ 

6.40 

6.40 

Vitamin mixture 

0.50 

0.50 

DL-Methionine 

0.20 

0.20 

L-tryptophan 

0.04 

0.04 

Amino Acid mixture^ 

— 

4.60 


Solka floe. Brown Company, Berlin, N.H. 


Minerals in g/kg diet: CaHP0 4 H 2 0, 18.04; CaC0 3 , 19.04; KH 2 P0 4 , 14.03; NaHC0 3 , 
8.82; MnS0 4 H 2 0, 0.35; FeS0 4 H 2 0, 0.50; MgSO 4 ,3.02; KI0 3 , 0.01; CuS0 4 -H 2 0, 
0.03; ZnC0 3 , 0.15; CsC1 2 -6H 2 0, 0.002; NaMo0 4 H 2 0, 0.008; and Na 2 Se0 3 , 0.002. 

The vitamin mixture included 10,000 IU Vitamin A as retinyl palmitate; 4,000 IU 
Vitamin D as Cholecalciferol; 75 IU Vitamin E as dl-a-tocopheryl acetate; and the 
following amounts of vitamins in mg/kg diet: thiamin'HCL, 40.0; menadione 
dimethylpyrimidinol bisulfite (Vitamin K), 2.0; riboflavin, 30.0; D-calcium 

pantothenate, 150.0; niacin, 300.0; pyridoxine'HCL, 20.0; d-biotin, 0.5; folic acid, 
15.0; Vitamin B^, 0.3; Ethoxyquin (100%), 200.0; and myo-inositol, 500.0. 

Amino acids in g/kg diet: L-threonine, 7.0; L-valine, 5.0; L-cystine, 3.0; L-lsoleucine, 
10.0; L-leucine, 8.0; lysine'HCL, 2.0; and L-arginine'HCL, 11.0. 


Lipid Extraction and Analysis 

Samples were collected, weighted, measured, lyophilized, and stored at 
-20°C under nitrogen. Several small fish or approximately one gram of each 
diet for each sample were rehydrated with 5 ml distilled water. Samples were 
extracted twice in a Sorvall Omni-mixer (60 ml capacity), according to the 
Bligh and Dyer (11) technique as modifed by Kates (23). Lipids were 


219 





determined gravimetrically. The lipid material was saponified with 10 ml 0.5 N 
potassium hydroxide-methanol. 

Fatty acids were methylated with 14 percent boron trifluoride-methanol 
(28). Fatty acid methyl esters (FAME) were injected into a single column 
Varian Aerograph 1200 gas-liquid chromatography unit operated isothermally 
at 180°C and equipped with a flame ionization detector. FAME were separated 
on a 15 percent diethylene glycol succinate (DEGS) column, on 100-120 mesh 
Chromosorb W-HP, 2.1 m long x 3.2 mm O.D., supplied with 75 ml/min flow 
of nitrogen as the carrier gas and a three percent ethylene glycol succinate 
polyester-Z (EGSP-Z) column (same dimensions as DEGS) on 100-120 mesh 
Gas Chromosorb Q support with 40 ml/min nitrogen. Indentification and 
quantification of the FAME were made with an electronic integrator (Hewlett 
Packard 3380A) supplied with the relative retention times of authentic 
standards and literature values for published oils (2). Cod liver oil was used as a 
secondary standard (3) and heptadecanoic acid (17:0) was used as an internal 
standard (16). Unresolved chromatogram peaks were detected by comparing 
the profiles of the two individual column separations. 

RESULTS AND DISCUSSION 

Artemia Diet—Batch Culture 

The total protein levels and amino acid profiles are given in Table 15-4 for 
the wild silversides, their eggs, and laboratory cultured fish of various ages. The 
cultured fish had been fed the live 3-day-old brine shrimp diet. The amino acid 
spectrum of the silversides was very similar to the spectrum of migrating 
Atlantic salmon (23). 

The brine shrimp analysis was similar to the results of Gallagher and Brown 
(18) who also analyzed San Francisco Bay brine shrimp. These authors stated 
that methionine in the brine shrimp may be limiting compared to standard egg 
albumin levels. However, our results showed that the methionine levels in brine 
shrimp were very similar to the level found in the silverside eggs. The major 
differences between the 3-day-old brine shrimp and the silverside eggs were the 
lower levels of threonine, serine, proline, valine, and leucine, and the higher 
levels of arginine in the brine shrimp. 

The silverside eggs contained higher levels of threonine, serine, proline, 
alanine, leucine, and tyrosine, and lower levels of glycine and methionine than 
were found in the wild fish. The amino acid profile of the 5-day-old silversides 
changed substantially from the profile of the eggs. Most of the changes resulted 
in a general decrease in amino acids from the egg to the larval stage. 


220 


Table 15-4. Amino Acid Profiles of Silversides, 
Expressed as Gram Amino Acid Per 100 Gram Protein 


Amino Acid 

Cultured Silversides 

Brine 

Wild e 99s Shrimp 

Silversides Unfert. 5-day 10-day 25-day 58-day Diet 

Aspartic Acid 

9.3 8.7 10.2 

9.9 9.9 

10.2 8.9 

Threonine 

4.3 6.4 4.6 

5.1 4.7 

4.4 3.1 

Serine 

4.3 8.2 4.9 

4.9 5.2 

4.6 4.4 

Glutamic Acid 

14.1 13.7 15.2 14.4 15.2 

13.8 11.6 

Proline 

3.9 7.5 4.2 

4.3 4.5 

4.8 3.1 

Glycine 

6.0 3.7 5.8 

6.0 6.4 

6.6 4.3 

Alanine 

6.0 7.7 4.7 

4.5 4.9 

6.2 6.0 

Valine 

5.3 6.8 5.5 

5.6 5.8 

5.6 5.1 

Methionine 

3.6 2.1 

1 2.5 

2.8 2.2 

3.3 2.3 

Isoleucine 

4.6 5.9 4.8 

5.1 4.4 

4.0 4.9 

Leucine 

7.7 10.6 7.7 

7.8 7.2 

7.5 8.9 

Tyrosine 

3.5 5.1 

1 3.9 

4.0 3.9 

3.2 4.2 

Phenylalanine 

4.2 4.7 4.7 

4.6 4.6 

4.3 4.5 

Histidine 

3.6 2.9 2.9 

2.9 2.7 

5.1 1.2 

Lysine 

8.9 8.3 8.3 

8.7 8.3 

8.9 9.2 

Arginine 

6.3 7.1 

1 7.3 

7.1 7.3 

6.5 10.5 

% Protein 1 

15 NA‘ 

2 13 

12 NA 

14 NA 

% Moisture 

NA 84 

79 1 

B1 81 

79 NA 

1 wet weight basis 





2 

NA = not available 





Tryptophan was not determined by the procedure used in this study. 




Table 15-5. Fatty Acid Composition of Unfertilized Eggs 



and two 15-Day-Old Silversides Fed 3-Day Old Brine Shrimp Nauplii 



Wild 

2-Day-Old 

15-Day-Old 


Unfertilized 

Silversides 

Sac fry (6.0 mm) 

Silversides 

Fatty Acids 

Eggs 

(20.5 mm) 

Silversides 

(11.0 mm) 

14:0 

2.50 1 

1.34 

1.14 

0.69 

14:1 

0.27 

0.14 

0.06 

0.30 

15:0 

0.77 

0.55 

0.36 

0.33 

15:1 

0.15 

0.14 

0.02 

0.19 

16:0 

18.67 

22.55 

22.67 

16.29 

16:1 

7.40 

5.83 

4.48 

10.06 

17:1 

1.28 

0.14 

0.13 

1.49 

18:0 

5.99 

9.44 

9.74 

7.34 

18:1cu9 

14.19 

10.40 

12.83 

25.33 

18:2co6 

1.58 

1.23 

0.68 

2.24 

18:3to6 

0.55 

0.38 

0.32 

0.52 

1 8:3cj3 

1.47 

0.88 

0.44 

2.49 

1 8:4cj3 

0.88 

0.75 

0.47 

0.34 

20:1cj9 

1.36 

0.71 

0.20 

0.49 

20:4cj6- 

2.71 

3.75 

2.52 

5.13 

20:5oo3 

8.09 

7.37 

4.90 

7.84 

22:5cu3 

3.72 

1.34 

2.33 

3.26 

22:6cj3 

27.15 

35.53 

36.05 

14.96 

% oil 2 

13.9 

8.5 

NA 

NA 

cu3/tu6 Ratio 

8.5 

8.0 

12.6 

3.7 


weight percent 

2 

based on dry weight 


221 








From the Fifth to the 58th day of culture, the amino acid profiles did not 
change markedly. The only changes which occurred were a decrease in glutamic 
acid and an increase in alanine and histidine. Compared to the 3-day-old brine 
shrimp diet, the 58-day-old fish differed only in the levels of histidine and 
arginine. Therefore, it seems that the dietary amino acids were absorbed and 
deposited as early as the fifth day of life. 

Table 15-5 shows the fatty acids of unfertilized silverside eggs and 2 and 
15-day-old fry fed on 3-day-old brine shrimp nauplii. The unfertilized eggs had 
a whole body lipid level of 13.9 percent and the fatty acids 20:5co3 and 
22:6co3 comprised more than 35 percent of the total fatty acids. The co3 acids 
exceeded the co6 component by greater than eight times. It would appear that 
the high energy level coupled with the large co3 polyunsaturated fatty acid 
(PUFA) component are indicative of their metabolic and physiological 
importance in the early life stages of silversides. 

In the 2-day-old yolk sac fry the acids 16:0, 18:0 and 22:6co3 were 
preferentially retained from the energy rich egg, while 16:1, 18: lco9, 20:5co3 
and those acids which comprise individual contributions of less than 4 percent 
each showed reduced levels. The co3/co6 ratio of the 2-day-old yolk sack fry 
increased to 12.6, from the egg level of 8.5. A similar pattern of fatty acid 
retention and utilization was found by Hayes (19) and his associates in the 
total lipids of developing steelhead trout, Salmo gairdneri. 

The brine shrimp diet was composed largely of 16:0, 16:1, 18:lou9, and 
20:5oo3 but contained no 22:6co3 (Table 15-6). The analyses of silversides fed 
this diet (Tables 15-5 and 15-6) showed that the fatty acids 16:1 and 18:lco9 
increased from the 2-day-old yolk sac fry levels, while 16:0 and 22:6co3 
decreased. It is evident that the fish change their concentration of fatty acids 
to reflect the general composition of their diets. Other researchers have made 
the same correlation between the diet and tissue fatty acids of cultured fish (1, 
9,14, 23,24). 

In silversides cultured for 137 days (30) the level of 20:5co3 and 22:6co3 
represented as little as three percent of the total fatty acid composition. The 
level of these two fatty acids in the wild fish represent an amount about ten 
times this level. Additionally, the wild fish had an oil content of only about 
eight percent, whereas 137-day-old cultured fish had a lipid level of 21.4 
percent (30). Thus, brine shrimp fed fish did not closely resemble the lipid 
content of their natural counterparts. Since the co3 acids have been shown to 
play a chief role in the metabolism of fish, it would seem that the amount of 
lipid storage may be related to a certain minimal amount of to3 PUFA, namely 
22:6 gj 3. A mechanism may exist which enhances the absorption and 
deposition of lipids to ensure a minimal 22:6co3 tissue level. Therefore, the 


222 


Table 15-6. The Major Fatty Acids of the 3-Day-Old 
Brine Shrimp Diet and 25 and 58-Day-Old Silversides 



3-Day-Old 

25-Day-Old 

58-Day-Old 


Brine 

Juvenile 

Juvenile 


Shrimp 

Silversides 

Silversides 

FAME 

(Diet #2) 

(13.05 mm long) 

(22.24 mm long) 

16:0 

11.45 

16.77 

21.05 

16:1 

16.49 

8.35 

12.76 

18:0 

4.10 

8.72 

9.19 

18:1 co9 

34.34 

27.44 

36.87 

1 8:2co6 

4.78 

2.36 

3.19 

1 8:3co3 

4.67 

2.85 

2.26 

20:1 co9 

0.55 

0.56 

0.70 

20:4to6 

3.13 

5.99 

3.79 

20:5co3 

13.31 

7.89 

3.87 

22:5co3 

— 

2.23 

1.73 

22:6co3 

— 

13.67 

1.97 

% oil 

10.00 

12.90 

12.40 

co3/eo6 ratio 

2.27 

3.19 

1.41 


3-day-old brine shrimp diet may lead to critical nutritional problems if used in 
a long term study. 

When compared to the wild fish (Table 15-5), the cultured fish have a far 
lower co3 fatty acid level and much higher level of the co6 acids. The co3/co6 
ratio of the wild fish lipid was 8.0, more than two times the cultured fish 
levels. The wild fish fatty acid profile was similar to the egg and 2-day-old yolk 
sac fry values, as would be expected. In the wild fish the fatty acids 20:5co3 
and 22:6co3 represented about 40 percent of the total fatty acid composition. 
Preferably, the cultured fish should resemble the wild juvenile fish in our 
experiments. 

Artificial Diets 

Since the amino acid profiles of the Atlantic salmon and wild silversides 
were comparable, a commercial salmon diet was tried in the preliminary 
evaluation of the artificial diets. Compared to the brine shrimp fed fish, growth 
and survival in the test diets fed group was very poor. Fish on the salmon type 
diet exhibited some scoliosis. Two factors which could have contributed to this 


223 






problem were the leaching of dietary components from the artificial diet when 
it became water soaked, or perhaps an inadequate lipid composition (soybean 
oil). In reference to the latter point, the fatty acid composition of the artificial 
diet is shown in Table 15-7 along with the spectrum for soybean oil and that of 
silversides cultured on the salmon type diet. The soybean oil diet and the oil 
resemble each other to some degree, since 16:0, 18:lco9, and 18:2 oj 6 are the 
major fatty acids of both analyses. Likewise, silversides fed this artificial diet 
closely resemble the lipid make-up of the diet they were fed. However, it is 
evident that fish fed the artificial diet were not similar in fatty acid 
composition to the wild fish, and thus this diet had not accomplished the 
major goal of providing a laboratory cultured fish of similar biochemical 
composition to the wild fish (Table 15-5). 

Based on the biochemical analyses of the diets and cultured fish, and the 
results of poor growth and survival, the salmon diet was modified by the 
addition of a marine oil (cod liver oil). The soybean oil based diet was again fed 
to verify past results. In addition to these two diets, the live brine shrimp diet 
was used along with: a freeze-dried form of brine shrimp, a wild plankton diet, 
Tetra Marin—a commercial aquarium food, and two semi-purified diets with 
various amino acid compositions (Table 15-3). The cultured fish were again 
analyzed for protein and lipid composition. 

The growth and survival results of fish fed these experimental diets are 
presented in Table 15-8. The live brine shrimp diet gave the best survival (97 


Table 15-7. The Major Fatty Acids of Soybean Oil, 
an Artificial Diet (#7) with a Soybean Base, 
and Silversides Cultured on the Diet 


FAME 

Artificial Diet 

With Soybean Oil 

Soybean 

Oil 

Silversides Fed 

the Artificial Diet 
(13.97 mm long) 

16:0 

13.05 

12.52 

17.43 

16:1 

2.43 

— 

3.74 

18:0 

2.45 

4.69 

7.41 

18:1co9 

36.12 

19.25 

36.37 

18: 2cj6 

29.81 

54.90 

21.22 

18:3co3 

1.88 

7.83 

0.51 

20:1 co9 

2.94 

— 

2.95 

20:5co3 

1.62 

— 

— 

22:6co3 

2.08 

— 

4c 76 

go3/go3 

0.21 

0.20 

0.36 


224 







Table 15-8. Percent Survival and Weight Gains for Silversides 
Cultured on Various Experimental Diets 
(Modified from Beck and Poston (7))^'^ 



Survival 

Weight Gain 

Experimental Diet 

% 

mg 

% 

1. Wild plankton 

54.0 

4.3 

48.3 

2. Brine shrimp-live 

97.0 

36.6 

411.2 

3. Starved 

0.0 

— 

— 

4. Brine shrimp-dried 

66.0 

4.5 

50.6 

5. Tetra Marin 

95.0 

4.4 

49.4 

6. Artificial, HPM-1 

51.0 

0.6 

6.7 

7. Artificial, CHP-1 

28.0 

1.2 

13.5 

8. Artificial, MP-1 

65.0 

-0.7 

-7.9 

9. Artificial, MP-2 

61.0 

-0.5 

-5.6 


Diet #1 data only one replicate, diets 2, 4-9 are averages of two replicates. 

2 

Fish were 23 days old at onset of the study and were cultured for 23 days. 


percent) and the best weight gain (411 percent). Normal growth of the wild 
fish has been estimated at 12 mm per month during the growth period (6). 
None of the other diets gave an appreciable weight gain; in fact some groups 
actually lost weight. With the exception of Tetra Marin, all the artificial diets 
produced a relatively poor survival rate. 

Table 15-9 shows the fatty acid composition of the natural and artificial 
diets. The effect of the dietary fatty acids on the cultured fish lipids is 
presented in Table 15-10. The diet profiles and the respective cultured fish 
profiles were quite similar. The cultured fish fed the cod liver oil based diets 
(Diets 6, 8, 9) and those fed Diet 5 contained a much higher level of 22:6co3 
than the brine shrimp fed fish (Table 15-6 and 15-10) or the soybean oil fed 
fish (Table 15-7 and 15-10). These fish more closely resembled their wild fish 
counterparts. A lipid modification of the salmon diet had therefore effected a 
biochemical change in the fish to a more “wild like” laboratory fish. The 
replacement of the soybean oil in Diet 7 by cod liver oil (Diet 6) doubled the 
survival level, however growth was only one-half as great. It is very difficult to 
draw and direct correlation between the dietary lipid composition of the 
various diets and survival, since the experimental design of this study was quite 
unlike the classical nutrition experiments. 

The comparison of the amino acid profiles of fish fed the natural and 
artificial diets indicated little variation between the treatment groups (Table 


225 





Table 15-9. Percentage Composition of Fatty Acids from Total 
Lipids of the Various Experimental Diets 




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226 


co3/cj 6 ratio 6.88 2.16 2.66 0.41 2.65 0.21 6.27 6.81 







Table 15-10. Percent Composition of the Fatty Acids from Total Lipids of 
Silversides Cultured for 23 Days on the Various Experimental Diets. 



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% oil 12.88 11.27 19.90 10.79 11.72 12.82 NA 15.45 12.44 NA 

cj3/c*j 6 ratio 3.07 5.15 2.18 2.65 1.12 2.84 0.36 3.27 4.46 8.06 







15*11). Although histidine and methionine were present in greater amounts in 
Diets 6 and 9 than in the other treatment groups, no relationship is obvious 
between these amino acid levels and growth and survival. The higher amounts 
of leucine in the brine shrimp fed fish could indicate a role of this amino acid 
in their greater growth and survival. However, this was not evident in the 
freeze-dried brine shrimp fed group. 

No substantial differences were found in growth and survival of fish fed the 
nonsupplemented amino acid diet (Diet 8) versus the supplemented diet (Diet 
9). Therefore, it is difficult to say the quantity of dietary amino acids influence 
the metabolism and utilization of the various diets. Bioavailability studies will 
be necessary to ascertain the degree of incorporation of the supplemented 
amino acids. 

CONCLUSIONS 

1. The best growth and survival of juvenile silversides was obtained on a live 
3-day-old brine shrimp nauplii diet. Substantially lower growth and 
survival was obtained on the artificial diets. 

2. Freeze-dried brine shrimp provided less growth than a live brine shrimp 
diet. Live brine shrimp must contain some component which is removed 
or altered upon freeze drying. 

3. It is difficult to say whether protein (amino acids) in the diets was a 
factor in the differences in growth and survival of cultured fish fed the 
artificial diets and the brine shrimp fed fish. The brine shrimp fed fish 
did, however, contain higher levels of leucine than all other cultured 
groups. Bioavailability studies will be necessary to ascertain the degree of 
assimilation and incorporation of dietary amino acids. 

4. Whole body lipid fatty acid composition of cultured fish changed to 
reflect the composition of their diets. Fish fed the brine shrimp diet had 
higher fat levels and lower polyunsaturated fatty acid levels than wild 
fish. Cultured fish appear to be storing large amounts of lipids in order to 
obtain a threshold level of the polyunsaturated fatty acids. 

5. Fish fed a cod liver oil based diet more closely resembled their wild 
counterparts. However, growth and survival were poor compared to 
3-day-old brine shrimp fed fish. 

ACKNOWLEDGEMENTS 

The experimental part of the project required a close working arrangement 
between scientists of the marine fish culture team at the Environmental 


228 


Table 15-11. Amino Acid Profiles of Silversides Fed the Various Artificial 
and Natural Diets Expressed as Gram Amino Acid Per 100 Gram Protein 


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Protection Agency, under the direction of Mr. Allen Beck, and food scientists 
at the University of Rhode Island. This cooperative effort has promoted an 
investigation that neither group could have accomplished easily on an 
independent basis. 

This work was supported by the Environmental Protection Agency under 
Grant #R-803818 and the Rhode Island Agricultural Experiment Station. This 
manuscript is Rhode Island Agricultural Experiment Station Contribution 
Number 1766. 

Thanks is given to Allen Beck, Grace MacPhee, Bruce Lancaster and Drs. 
Robert Barkman and Hugh Poston for their contributions. Additionally, we 
thank the personnel at the Tunison Laboratory of Fish Nutrition for 
formulating and pelleting the artificial diets, and providing advice and guidance 
to the total project. Thanks is also given to Cindy Seidel for typing of the 
manuscript. 

REFERENCES 

1. Ackman, R.G. and C.A. Eaton, 1966. Some Commercial Atlantic Herring 
Oils: Fatty Acid Composition. J. Fish. Res. Bd. Can. 23: 991. 

2. Ackman, R.G. and R.D. Burgher, 1964. Cod Liver'Oil: Component Fatty 
Acids as Determined by Gas-Liquid Chromatography. J. Fish. Res. Bd. Can. 
21: 319. 

3. Ackman, R.G. and R.D. Burgher, 1965. Cod Liver Oil Fatty Acids as 
Secondary Reference Standards in the GLC of Polyunsaturated Fatty Acids 
of Animal Origin: Analysis of the Dermal Oil of the Atlantic Leatherback 
Turtle. J. Am. Oil Chem. Soc. 42: 38. 

4. Bardach, J.E., J.H. Ryther and W.O. McLarney, 1972. Aquaculture—The 
Farming and Husbandry of Freshwater and Marine Organisms. Wiley 
Interscience— John Wiley and Sons, Inc., New York, 868 pp. 

5. Barkman, R.C. and A.D. Beck, 1976. Incubating Eggs of Atlantic 
Silversides (Menidia menidia) on Nylon Screen. Prog. Fish. Cult. 38: 148. 

6. Bayliff, W.H., 1950. Life History of the Silverside (Menidia menidia 
Linnaeus). Chesapeake Biol. Lab., 90: 27 pp. 

7. Beck, A.D. and H.A. Poston. Effects of Diets on Survival and Growth of 
the Atlantic Silverside {Menidia menidia). Submitted to Aquaculture, Mar. 
1977. 


230 


8. Benijts, R., E. Van Voorden and P. Sorgeloos, 1975. Changes in the 
Biochemical Composition of the Early Larval Stages of the Brine Shrimp 
(Artemia salina L.) Proc. 10th European Symposium on Marine Biol., 
Ostend, Belgium, 1:1. 

9. Bishop, D.G., D.G. James and J. Olley, 1976. Lipid composition of Slender 
Tuna (Allothunnus fallai) as Related to Lipid Composition of their Feed 
(Nyctiphanes australis). J. Fish. Res. Bd. Can. 33: 1156. 

10. Blackburn, S., 1968. Amino Acid Determination: Methods and Techniques, 
Marcel Dekker, Inc., New York. 271 pp. 

11. Bligh, E.G. and W.J. Dyer, 1959. A Rapid Method of Total Lipid 
Extraction and Purification. Can. J. Biochem. Physiol. 37: 911. 

12. Buss, K., 1959. Jar Culture of Trout Eggs. Prog. Fish. Cult. 21: 26. 

13. Chibnall, A.C., M.W. Rees and E.F. Williams, 1943. The Total Nitrogen 
Content of Egg Albumin and Other Proteins. Biochem. J. 37: 354. 

14. Colvin, P.M., 1976. The Effect of Selected Seed Oils on the Fatty Acid 
Composition and Growth of Penaeus indicus. Aquaculture 8: 81. 

15. Cowey, C.B., K.W. Daisley and G. Parry., 1962. Study of Amino Acids, 
Free or as Components of Protein and Some B Vitamins in the Tissues of 
the Atlantic Salmon, Salmo salar, during Spawning Migration. Comp. 
Biochem. Physiol. 7: 29: 

16. Farrington, J.W., J.G. Quinn and W.R. Davis, 1973. Fatty Acid 
Composition o i Nephtys incisa and Yoldia limatula. J. Fish. Res. Bd. Can. 
30: 181. 

17. Fava, J.A. and J.W. Meldrin, 1977. Response and Tolerance of Atlantic 
Silversides to Chlorine. 33rd Northeast Fish and Wildlife Conference, 
Boston, Massachusetts, 3-6 April. 

18. Gallagher, M. and W.D. Brown, 1975. Composition of San Francisco Bay 
Brine Shrimp (Artemia salina). J. Agric. Food Chem. 23: 630. 

19. Hayes, L.W., I.J. Tinsley and R.R. Lowery, 1973. Utilization of Fatty 
Acids by the Developing Steelhead Sac-Fry (Salmo gairdneri). Comp. 
Biochem. Physiol. 45B: 695. 


231 


20. Hiller, A., V. Plazin and D.D. VanSlyke, 1948. A Study of Conditions for 
Kjeldahl Determination of Nitrogen in Proteins. J. Biol. Chem. 176: 1401. 

21. Hirs, C.H.W., 1967. Automatic Computation of Amino Acid Analyzer 
Data. In: Enzyme Structure: Methods in Enzymology, S.P. Colowick and 
N.O. Kaplan, Editors-in Chief; C.H.W. Hirs, Editor, Academic Press, New 
York, Vol. XI. 

22. Jefferies, H.P., 1970. Seasonal Composition of Temperate Plankton 
Communities: Fatty Acids. Limol. Oceanog. 15: 419. 

23. Kates, M., 1972. Techniques of Lipidology: Isolation, Analysis and 
Identification of Lipids. North Holland Publishing Company, New York, 
347. 

24. Lee, D.J., J.N. Roehm, R.C. Yu and R.O. Sinnhuber, 1967. Effect of co3 
Fatty Acids on the Growth Rate of Rainbow Trout (Salmo gairdneri ). J. 
Nutr. 92: 93. 

25. Lovern, J.A., 1950. Some Causes of Variation in the Composition of Fish 
Oils. J. Soc. Leather Tech. Chem. 34: 7. 

26. Middaugh, B.P. and P.W. Lempsis, 1976. Laboratory Spawning and Rearing 
of a Marine Fish, the Silverside Menidia menidia. J. Mar. Biol. 35 (4): 295. 

27. Moore, S. and W.H. Stein, 1963. Chromatographic Determination of 
Amino Acids by use of Automatic Recording Equipment. In: Enzyme 
Structure: Methods in Enzymology, S.P. Colowick and N.O. Kaplan, 
Editors-in-Chief; C.H.W. Hirs, Editor, Academic Press, New York, Vol. VI: 
819. 

28. Morrison, W.R. and L.M. Smith, 1964. Preparation of Fatty Acid Methyl 
Esters and Dimethyl Acetals from Lipids with Boron Trifluoride-Methanol. 
J. Lipid Res., 5: 600. 

29. Niederwieser, A. and G. Pataki, 1971. New Techniques in Amino Acid 
Peptide and Protein Analysis, Niederwieser and Pataki (eds.), Ann Arbor 
Science Publishers, Inc., Michigan. 73 pp. 

30. Schauer, P.S., 1977. Lipid Metabolism and Fatty Acid Composition of Wild 
and Cultured Atlantic Silversides (Menidia menidia). Masters Thesis, 
University of Rhode Island. 


232 


31. Spackman, D.H., W.W. Stein and S. Moore, 1958. Automatic Recording 
Apparatus for use in the Chromatography of Amino Acids. Anal. Chem. 
30: 1190. 

32. Sorgeloos, P., M. Baeza-Mesa, C. Claus, G. Vandeputte, F. Benijts, E. 
Bossuyt, E. Bruggeman, G. Personne and D. Versichele, 1977. Artemia 
salina as Life Food in Aquaculture. In: Fundamental and Applied Research 
on the Brine Shrimp, Artemia salina (L.) in Belgium. Spec. Publ. No. 2 
European Mariculture Society. 


233 


THE COMBINED EFFECT OF TEMPERATURE 
AND DELAYED INITIAL FEEDING OF THE 
SURVIVAL AND GROWTH OF 
LARVAL STRIPED BASS 
Morone Saxatilis (WALBAUM) 

Bruce A. Rogers 

Graduate School of Oceanography 
University of Rhode Island 
Kingston, R.l. 02881 

Deborah T. Westin 
Graduate School of Oceanography 
University of Rhode Island 
Kingston, R.l. 02881 


ABSTRACT 

Rearing temperature and the time of first feeding interact to determine the 
degree of survival and rate of growth in larval striped bass. Between 15 and 
27°C, temperature affects the rate of growth and development in fed groups, 
and the time to death by starvation in unfed lots. Delayed first feeding retards 
structural development. The ‘point-of-no-return’ in striped bass is very near the 
stage of complete mortality due to starvation. Unfed groups survived up to 22 
days after hatching at 24°C and 32 days at 15°C. Larvae fed late into 
starvation survived and continued to grow at a rate somewhat higher than that 
observed in earlier fed groups at all temperatures. Larvae which has survived 
delayed development were indistinguishable on the basis of external 
morphology from much younger individuals reared under more favorable 
conditions. The effects of nutritional and thermally induced developmental 
retardation are discussed in terms of how they may affect larval growth and 
mortality rate estimates used in assessing the effects of estuarine power plants. 

INTRODUCTION 

Many estuarine and marine fish species, including the striped bass, Morone 
saxatilis (Walbaum), produce large numbers of relatively small pelagic eggs at 
spawning. These smaller eggs contain fewer yolk reserves. After a relatively 
short incubation period, they hatch into prolarvae that are, in general, at a 
more rudimentary stage of structural development than those of species 


234 


producing fewer but larger eggs (3, 17). Development continues while the 
young larvae drift in the water column, absorbing their yolk and developing 
the mouth parts and swimming ability to capture food and avoid predators. 
Although the methods used to determine the extent of first year mortality in 
natural populations are at best imprecise (25), it is clear that among high 
fecundity species, losses early in life are extremely high, with the highest 
mortality rates among the early larval stages. 

As early as the end of the last century, Fabre-Domergue and Bietrix (9) 
encountered heavy mortality among laboratory reared marine Fish larvae which 
had exhausted their yolk reserves. Hjort (12) concluded, based on his studies of 
year to year fluctuations in Norwegian cod and herring abundance, that 
year-class strength was probably determined early in the larval development of 
these species. The term “critical phase” or “critical period” has been used, in a 
general sense, to refer to that span of time in the early development of the 
individuals comprising a particular year-class during which the ultimate number 
of recruits is determined (11). In a narrower usage “critical period” may be 
used to refer to that point in development of the larval fish at which all sources 
of endogenous (yolk) nutrition have been consumed, and active feeding must 
commence if death by starvation is to be avoided. Hjort (12) proposed death 
following yolk exhaustion as only one of several possible mechanisms by which 
events early in development might affect the subsequent size of a given 
year-class. In 1956, Marr (23) reviewed the available evidence in support of the 
existence of a “critical period”. He concluded that there was little evidence to 
suggest that mass starvation occurred in the sea among larvae that had recently 
absorbed their yolk, or that survival curves for natural populations revealed any 
noticeable inflection at the point of yolk absorption. 18 years later, May (25) 
noted that little new data has been gathered since Marr’s review that could 
contribute meaningfully toward the resolution of the problem of whether or 
not a “critical period” at yolk absorption exists as a widespread phenomenon 
among fish species. He suggested that while among high fecundity species, 
year-class strength is certainly determined during early development as Hjort 
maintained, the physiological mechanisms that have evolved to meet 
environmental challenges that confront the developing larva must be addressed 
on a species by species basis. 

The prolarva, from the time it is hatched until it captures its first meal, is 
reliant on its yolk reserves to provide the structural materials for continued 
ontogenetic development, as well as to provide energy to fuel its maintenance, 
activity, and growth needs. Unless sufficient satisfactory food is taken after the 
exhaustion of yolk reserves, structural tissue already laid down is metabolized 
to support the continued costs of swimming in search of prey, until the larvae 
is so debilitated by the effects of starvation that it is unable to capture and 
utilize suitable prey when it does become available. Blaxter and Hempel (4) 


235 


termed this ecological death-point the “point-of-no-return” (PNR). Starved 
larvae may live after the PNR has been reached but with no likelihood of 
ultimate survival. The time span between the development of the ability to 
feed and PNR determines how important the period of transition to exogenous 
feeding will be to the survival of larvae of a particular species. 

The rate of growth and development of larval fish is very much temperature 
dependent. Among the studies in which the relationship between rearing 
temperature and larval growth rate have been demonstrated are those of 
Kramer and Zweifel (16), Houde (14), and Shelbourne et al (33). In all these 
studies, larval growth rate increased with increasing temperature, except where 
survival limits were approached. 

Weight specific metabolic rate (oxygen consumption) also increased with 
increasing temperature among fish larvae of the same weight in studies such as 
those of Holiday et al (13) and Laurence (20). 

The striped bass, Morone saxatilis (Walbaum), is a commercially important 
anadromous teleost native to the Atlantic coast of North America. The natural 
range of the striped bass extends along the Atlantic coast of North America 
from the St. Lawrence River to Louisiana, with its center of abundance 
between Cape Cod and Cape Hatteras (26). There have been many introduced 
populations ranging from the extremely successful Pacific coast estuarine 
population, introduced in the 1880’s, to the many landlocked populations 
which have been established in natural and man-made freshwater 
impoundments in the southeastern states. 

Sexually mature striped bass enter and ascend rivers to the spawning 
grounds within the period between March and July. Peak spawning generally 
occurs at a water temperature on the spawning grounds of 15 to 18°C. 
Spawning sites are typically well into the freshwater portion of the estuary, 
although often within the range of tidal influence (35). Each ripe female may 
produce from one to three million eggs. Newly shed eggs are 1.28 to 1.38 mm 
in diameter. Upon water hardening they swell to a diameter of approximately 
3.0 mm. Newly hatched larvae average 3-4 mm in length, and have a large yolk 
sac and oil globule (22). For several days after hatching, young prolarvae spend 
much of their time in a vertical, head-up position drifting in the current. Larvae 
develop functional mouth parts and are capable of feeding within 2-10 days 
after hatching at normally encountered river temperatures. Yolk is generally 
completely absorbed by the time the larva reaches 6 mm in length. 
Metamorphosis into essentially adult form occurs by the time the larvae are 
approximately 17 mm in length, generally 2 to 3 weeks after hatching. Feeding 
larvae are capable of consuming relatively large organisms as their first food. 
Planktonic Crustacea and their developmental stages predominate in their diets 
through most of their first year (27, 10). 


236 


The striped bass is a well-studied species and there is a voluminous literature 
relating to its biology (30). Relatively little attention, however, has been paid 
to the ecology of early life stages, the period during the life of the animal 
which is most important in the determination of year-class strength. Mansueti 
(22) presented descriptions of the eggs and larvae from collections from the 
Roanoke and Patuxent Rivers, and provided observations on the feeding and 
early growth of larvae in captivity. Doroshev (8) reviewed aspects of egg and 
larval development and added anecdotal observations on metabolic rate and 
graying rates of larvae. Bayless (2) provided a manual of culture methods as 
practiced in South Carolina hatcheries, but did not give many details of larval 
requirements beyond the yolk sac stage. Short term lethal temperature levels 
for eggs and larvae were presented by Albrecht (1), Davies (7), Shannon and 
Smith (32), Shannon (31), and Morgan and Rasin (28). Observations of prey 
selectivity among late larvae were reported by Meshaw (27) and Gomez (10). 
Daniel (6) presented data on the effect of food density on larval survival. 

In many spawning rivers on the Atlantic coast, major conflicts have arisen 
over the effect of power plant operations on striped bass recruitment, as a 
result of the entrainment of eggs and larvae in cooling water intakes, and later 
the impingement of juveniles on intake screens. Entrainment losses are highest 
among striped bass under approximately 3 cm in length. The duration of the 
period of major entrainment losses is a direct function of the time required for 
young bass to develop from semiplanktonic eggs to early juveniles large enough 
to escape intake currents. An assessment of plant impact must take into 
account the duration of entrainable life stages. To date, only crude estimates 
have been used in plant impact models involving striped bass (e.g., 21,36). 

The purpose of the present study was to determine in what way 
temperature and an initial delay in the onset of active feeding work together to 
affect the rate of survival and growth of striped bass larvae. Temperature is a 
controlling factor which may be expected to have a profound effect on the 
metabolic demands of the developing larva. The availability of food determines 
the extent to which these demands can be met. Temperature and delayed first 
feeding may be expected to interact in a manner which would largely 
determine the life span and early growth trajectory of the developing larva. By 
observing how water temperature and feeding level affect growth, better 
predictions of stage duration, hence vulnerability to power plant entrainment, 
may be made. 

MATERIALS AND METHODS 
Source of Study Material 

Eggs from Maryland used in the 1976 experimental series were netted from 
the Nanticoke River during the spawning season, using a 1 x 2 meter, 947 


237 


micron mesh neuston net. Eggs were also obtained from a striped bass hatchery 
run by the state of South Carolina at Moncks Corner, South Carolina. Eggs 
were air-shipped to the University of Rhode Island, where all experiments 
reported here were performed. 

Experimental Procedures 

For the duration of the relatively short incubation period, eggs were 
maintained in static 208 liter polyethylene drums filled with dechlorinated tap 
water. Best hatching success was observed when bacteria were controlled using 
an antibiotic. The antibiotic dosage used was 50,000 I.U./liter penicillin G plus 
50 mg/liter streptomycin sulfate. A strong air stream maintained the eggs in 
suspension and maintained an adequate dissolved oxygen level. Dead eggs 
floated to the surface and were removed as they were discovered. One-half of 
the volume of the tanks was replaced daily. Water temperature was maintained 
at laboratory room temperature, 14-16°C, during incubation. 

The experimental containers used in growth experiments consisted of four 
liter glass beakers. Prolarvae were stocked into these containers usually within 
24 hours of the time they were hatched. Larvae stocked at yolk absorption 
were held in their incubation containers until visible vestiges of yolk had 
disappeared. At stocking, all were of the same chronological age and had been 
exposed to the same conditions prior to the beginning of the experiment. No 
antibiotic was used in larval growth or survival experiments. The water used in 
all experiments was raised to 5°/oo salinity by mixing dechlorinated tap water 
with seawater (32°/oo, which had been passed through a cartridge filter rated 
to retain particles larger than 5 microns. Water in each container was changed 
every two days. 

Constant temperatures of 15, 18, 21, 24, and in some cases 27°C, were 
maintained in the test containers by keeping them immersed in temperature 
controlled water baths. Bath temperatures were controlled using Haake (model 
E-52) 1000 watt heater-thermoregulators operating against a cooling coil in 
each bath. Temperature excursions of no more than 0.25°C were normally 
encountered. The temperatures used span the range that might be encountered 
by developing larvae in nature. Bath temperatures were monitored on a 
strip-chart recorder and measured manually at least twice a day during the 
course of experiments. 

In initial experiments, dissolved, oxygen, pH, ammonia, and salinity 
measurements were made regularly. Dissolved oxygen was determined using the 
Y.S.I. D.O. probe, supplemented periodically with determinations using the 
azide-modification of the Winkler titration. The pH was measured using an 
Orion pH electrode. Ammonia was determined using a micro-modification of 


238 


the indophenol technique of Solorzano (34). Salinity measurements were made 
using an American Optical salinity refractometer. With frequent water changes, 
most of the water quality parameters changed insignificantly during the course 
of each experiment. In later experiments where the number of individual 
treatments became unmanageable, regular monitoring of all variables except 
temperature was discontinued, and a stringently maintained schedule of water 
changes observed. 


Feeding larvae were supplied with newly hatched Artemia nauplii at least 
twice a day in quantities sufficient to permit a portion to remain until the next 
feeding. Artemia nauplii proved to be a satisfactory diet for striped bass 
through the early juvenile stage. 

Larval growth was measured in terms of dry weight. Prior to weighing, 
larvae were lifted on a No. 6 sable brush, dipped in distilled water to remove 
any adherent salt or debris, blotted on filter paper and placed on a tared 
weighing pan. Pans were cut out of aluminum foil with a paper punch and 
ashed 4 hours at 500°C before use. Dry weight determinations were made after 
the specimen on its tared pan had been dried to a constant weight in a heated 
vacuum desiccator at 80°C over silica-gel. On larvae up to metamorphosis, 
weights were determined using a Cahn “Gram” electrobalance. Weights were 
read to the nearest microgram. 

Design of Experiments 

Temperature-delayed feeding-survival experiments were performed during 
the springs of 1976 and 1977. A total of three experiments were performed, 
each identical in its design. In each, five containers were placed in each of four 
constant temperature water baths. Each container was stocked with 100 
prolarvae at the temperature at which they had been held prior to the 
beginning of the experiment. Stocked containers were assigned to particular 
temperature treatments by lot. The acclimation period from holding 
temperature to the experimental temperature treatment was at most one hour. 

In each temperature treatment, the larvae in one container were offered a 
diet of newly hatched live Artemia nauplii at the beginning of the experiment. 
Food was withheld from another container throughout the observation period. 
The time of first feeding for larvae in each of the remaining three containers of 
starved larvae in each temperature treatment was determined on the basis of 
observations of the apparent state of health of individuals in each population. 
Each container was checked for mortality several times each day throughout 
each experiment, and all dead larvae removed. 


239 


Similar stocking and treatment procedures were used in 
temperature-delayed feeding-growth experiments. Food was withheld from one 
group at each temperature, and one group at each temperature was given food 
at the beginning of the experimental period. Initial dry weight measurements 
were made on a sample of 20 larvae at the beginning of the experiment. At the 
time each group was given its first food, a sample of 10 larvae from the unfed 
lot was weighed. At the end of the observation period all of the larvae in each 
treatment were measured and weighed. In cases where an intermediate growth 
observation was made between the time of first feeding, and before the end of 
the experiment, a sample of 10 larvae was used to establish growth of the 
population to this point. 

RESULTS 

Figure 16-1 shows the effect of delayed initial feeding on groups of larvae 
maintained at four temperatures. The survival time of the unfed control 
decreased with increasing temperature. The time to 50 percent mortality for 
unfed groups was 19, 21, 25 and 27 days after hatching among groups 
maintained at 24, 21, 18 and 15°C, respectively. Survival among early fed 
groups was generally highest in each temperature treatment. Among groups in 
which food was provided for the first time after up to 50 percent of the 
population had died, a portion of those remaining alive survived through the 
end of the observation period. A “point-of-no-return” beyond which survival 
could not occur even when food was provided, was nowhere in evidence in 
these experiments. In each temperature treatment the longer food was 
withheld, the greater the total mortality each group suffered. In cases where 
some additional mortality was observed after food was presented, there was 
generally evidence that the dead larvae had captured at least some food before 
expiring. 

Figure 16-2 shows the result of an experiment in which initial feeding was 
progressively delayed in a series of experimental groups of larvae held at five 
test temperatures. Changes in dry weight were used here to measure the rate of 
growth or shrinkage in fed and unfed groups at each temperature. Among 
starved lots, longevity increased and the rate of weight loss decreased at lower 
temperatures. Growth in dry weight increased rapidly once food was provided. 
In general, the effect of delayed initial feeding was to defer the attainment of a 
temperature-specific rate of growth of which larvae fed to satiation were 
capable. 

Larvae receiving their first food at day six after hatching at 15°C, had 
scarcely recovered their initial weight at hatching by the end of the 25-day 
observation period. Other groups which received their first food at day six after 


240 



o 

z 

X 

o 

< 

X 


X 

UJ 


< 


in 

> 

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Q 


IVAIAanS ±N3DU3d 


Figure 16-1. The Effect of Delayed Feeding on the Survival of 
Striped Bass Stocked at Yolk Absorption at 24, 21, 18 and 15°C. 

NOTE: Initial population 100 larvae. Numbered arrows indicate the order and 
time of first feeding. 


241 






























DAYS AFTER HATCHING 


Figure 16-2. The Effect of Temperature and Delayed Feeding on 
the Growth in Dry Weight of Striped Bass Larvae Stocked at 
Hatching at 27, 24, 21, 18 and 15°C. 

NOTE: Each sample contains 10 individuals. Numbered arrows indicate time 
of first feeding for each population. Symbols identify groups which received 
their first food at the same time. The location of symbols denotes sample 
means. Vertical bars indicate range of lengths in each sample. 


242 




























hatching, attained a mean dry weight of 0.5 mg at 25, 19, 18 and 12 days after 
hatching in temperature treatments of 18, 21,24 and 27°C, respectively. 

There was a tendency for later fed groups at each temperature to grow at a 
greater rate than those receiving their first food earlier in the experiment. Table 
16-1 shows the instantaneous growth coefficients calculated for delayed 
feeding groups at each temperature, using the same data that were presented 
graphically in Figure 16-2. In each case, the growth rates of the earliest fed 
groups were lower than those of groups which had been starved before 
receiving their first food. This growth compensation seldom permitted later fed 
groups to overtake those fed earlier, but did serve to partially offset the growth 
setback that resulted from later initial feeding. 

An additional effect of delayed initial feeding was a retardation of structural 
development which was observed at all temperatures. Figure 16-3 shows an 
example of the degree of developmeantal retardation which may occur among 
larvae of the same chronological age as a result of a delay in the timing of 
initial feeding. Among developing larvae, each developmental event appeared to 
coincide with the attainment of a particular larval length or dry weight. As a 
result, factors like temperature and nutritional state have a marked effect on 
the degree of structural development larvae of a particular age may achieve. 

DISCUSSION 

Among the fish species that have been investigated in the past, there appear 
to be several alternative patterns of survival following the delayed initial 
feeding of larvae which have consumed most or all of their yolk reserves. The 
northern anchovy, Engraulis mordax (Girard) (19) and the herring, Clupea 
harengus (4), both may survive food deprivation to a point beyond which 
continued survival is possible but ultimate recovery is not. The grunion, 
Leuresthes tenuis (Ayres) (24), on the other hand, can recover from food 
deprivation nearly up to the point of death through starvation. Observations 
reported here using striped bass and those of May (24) using grunion, are very 
similar. For neither species are the concepts of a “point-of-no-return” or ot a 
“critical period” at yolk absorption appropriate. In the striped bass, as in the 
grunion, protracted food deprivation results in a suspension of further 
structural development, and a gradual reduction in dry weight during starvation 
as the costs of continued maintenance are met at the expense of body tissues. 

The experiments of May (24) was performed at one temperature. In this 
study a range of temperatures was used. Temperature has been repeatedly 
shown to have a controlling influence on the rate of growth of fish larvae 
maintained on unlimited rations (e.g., 14, 16, 20). Similarly, temperature 
affects the rate of weight loss during starvation (18). Data presented in Figure 


243 


Table 16-1. Instantaneous Growth Coefficients for Delayed 
Feeding Groups at Five Constant Temperatures. 


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Figure 16-3. The Effect of Temperature and Delayed Feeding on 
the Growth in Dry Weight of Striped Bass Larvae Stocked at 

Hatching at 21 °C. 

NOTE: Each sample contains 10 individuals. Numbered arrows indicate time 
of first feeding for each population. Symbols identify groups which received 
their first food at the same time. The location of symbols denotes sample 
means. Vertical bars indicate range of lengths in each sample. Developmental 
twenty-one days after hatching is shown for larvae receiving their first food on 
days six and fourteen. 


245 
















16-2 of this study indicate that at lower temperatures the differences in size 
between early-fed and starved larvae was not great even three weeks after 
hatching. At higher temperatures both the rate of weight loss in starved 
populations, and the rate of weight gain in early fed populations increased 
markedly, with the major effect being seen on the rate of individuals in groups 
which were fed an unrestricted ration shortly after yolk absorption. 

There are vast differences between the conditions that exist in the 
laboratory and those in the natural habitat of the striped bass. These 
differences limit, to some extent, use of laboratory observations as an aid in 
interpreting conditions in the field. In these studies the only measurable 
mortality was that most closely associated with the availability of food. Losses 
due to predation, probably the most important sources of mortality in nature 
(5), were not involved at all here. It has frequently been suggested that the 
most likely victims of predation in nature might be individual larvae that have 
been weakened by the effects of starvation (5,11,15). 

Experimental groups in this study which received food were fed to excess. 
Therefore, the difference in growth attainment between starved and fed groups 
was probably at a maximum. Under conditions of restricted prey density, even 
larvae fed early in development might not have enjoyed as great a growth rate. 
At the same time, satisfactory food is probably never totally absent in nature 
as it was among the starved groups in this study. 

Artemia nauplii are a frequently used laboratory diet for the larvae of fish 
species that appear to require live food. Although Artemia nauplii appear to 
support a satisfactory rate of growth in laboratory populations, there is little 
nutritional information available to serve as a basis for comparison between 
Artemia and the variety of micro-crustacea that comprise the natural food of 
striped bass larvae (27). 

In nature, striped bass larvae are present on their estuarine nursery grounds 
during the spring under conditions of rapidly rising water temperatures. An 
average temperature rise of 1°C per week is typical in the Hudson River 
estuary during the period of larval striped bass abundance (36). Constant 
temperatures were used in these laboratory studies. 

With these reservations in mind, some statements may still be made about 
the probable early growth pattern of striped bass larvae under natural 
conditions. Data presented here indicate that the size and developmental stage 
of early striped bass larvae of a given chronological age are intimately related to 
their thermal and nutritional history. In well studied estuaries, the probable 
temperature history of a group of larvae spawned at a particular time and 
location may be estimated with some accuracy. However, a basis for 


246 


determining the nutritional history of a given group of larvae is not readily 
obtainable. Even where data are available on the spacial distribution and 
density of potential food organisms, the frequency with which larvae actually 
encounter suitable prey can never be known with any degree of accuracy (11). 

In assessing the effects of power generating plants on striped bass 
populations, it is necessary to estimate the rates of natural and plant induced 
mortality among the pelagic larvae. Life-stage duration estimates, coupled with 
estimates of stage-specific vulnerability to plant entrainment, may be used to 
determine the extent of losses that may be attributed to the operation of a 
particular plant. 

Larval mortality rates in nature are frequently estimated on the basis of the 
relative frequency of occurrence of larvae of various presumed age-classes in 
ichthyoplankton collections made throughout the period of larval abundance 
in the water column. The results of this study suggest that the occurrence of 
large numbers of early post yolk sac larvae in such collections may be a 
reflection of a period of suspended or slowed growth among larvae which are 
being subjected to heavy competition for the available food. Without some 
knowledge of hatching time, temperature regime, and feeding history, there 
appears to be no way that such larvae may be aged accurately on the basis of 
size and/or structural development alone. 

The use of fixed stage duration estimates in predictive models, especially for 
that stage immediately following yolk absorption, could lead to serious errors 
in the resulting estimates of stage-to-stage mortality rates. 

ACKNOWLEDGEMENTS 

This study was performed while under contract to the U.S. Environmental 
Protection Agency (Contract #68-03-0316). The authors wish to thank Janice 
Steele for typing the manuscript, and Margaret Leonard for drafting the 
figures. 


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Docket No. 50-286. Volume I and II. 


250 


THE EVOLUTION OF THE BUGSYSTEM: 
RECENT PROGRESS IN THE ANALYSIS OF 
BIO-BEHAVIORAL DATA 


Robert S. Wilson 
Department of Biology 
Yale University 
New Haven, Conn. 06520 

John O. B. Greaves 
Electrical Engineering Department 
Southeastern Massachusetts University 
North Dartmouth, Mass. 02747 


ABSTRACT 

Experimental investigation of the movements of organisms often entails the 
acquisition and processing of large samples of spatio-temporal data. An 
interactive, interpretive, on-line computer-television system (viz., the 
Bugsystem) was developed in order to expedite such analyses. Aspects of the 
structure of this prototype system are outlined. Its effectiveness is evaluated 
with regard to the problems confronting the bio-behavioral researcher. 

A second generation system has been developed under a research grant from 
the Environmental Protection Agency. Utilizing new hardware and software, it 
in many ways constitutes a generalization of its prototype. We describe 
features of the refined system which provide for the following: a large degree 
of machine-independence significant expansion of the size of data records, 
inclusion of experimental parameters and variables within the data structure, 
investigation of rotational and flectional movement, statistical analysis, and 
tracking of organisms in three dimensional space. Current utilization of the 
Bugsystem for research in behavioral physiology and the potential for future 
development are discussed. 

INTRODUCTION 

The fundamental focus of behavioral research is the description and 
explication of what individual organisms do. Because those biological activities 
most often classified as “behavior” consist largely (although not exclusively) of 
the movements of organisms, quantitative investigation of behavior is often 


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dependent upon the collection of temporal sequences of spatial information. 
Cinematography is the classical method used to gather such information. 
Behavioral variables such as the position of an organism, its orientation, and 
angles of flection of its appendages are extracted from the motion pictures by 
means of frame-by-frame analysis. An important advantage of this technique is 
its flexibility; it may, in principle, be employed in the investigation of any 
overt behavior which can be photographed. However, two factors preclude the 
widespread use of this method: (1) manual quantitization via frame-by-frame 
analysis is a lengthy and tedious process; and (2) once the data is obtained, a 
substantial amount of subsequent data processing may be required in its 
analysis. 

The Bugsystem (2, 4, 5) was designed to enable the acquisition and 
processing of video data by a minicomputer. Behavioral data are initially 
recorded using standard closed-circuit television equipment. These data are 
analyzed by replaying a video tape into a specially designed video-to-digital 
processor (christened the “Bugwatcher”). This device acts as an edge detector, 
greatly reducing the information flow to the computer and thereby allowing 
the real-time collection of spatial coordinates delineating the outlines of 
moving organisms. Frame-by-frame analysis of digitized video data is achieved 
through the use of specially designed programs tailored to the task of 
quantizing behavioral variables of interest to the researcher. 

The original version of the Bugsystem was developed by Greaves and 
implemented on an IBM 1800 computer at the University of California, Santa 
Barbara. This prototype system, previously described by Greaves (5), has been 
utilized by Hand and Schmidt (6) and by Wilson (8) to investigate the 
photokineses and phototaxes of marine dinoflagellates. However, several 
features of this system severely limited the domain of its application. 
Supported by a research grant from the Environmental Protection Agency, we 
have developed a second generation Bugsystem. Our explicit goal in the design 
of this system was to provide a flexible tool for the quantitative investigation 
of behavior, a system capable of realizing much of the potential of 
frame-by-frame analytic techniques. 

The purpose of this paper is to describe this second generation Bugsystem, 
emphasizing the way in which certain hardware and software refinements have 
expanded the scope of questions which may be conveniently answered by 
means of “bugwatching.” We discuss the way in which the user interacts with 
the system via a specially formulated “Behavioral Research Language” and the 
way in which this language has been implemented upon physical machines. We 
also describe the input of data to the system, the processing and display of 
behavioral data, and a variety of experimental strategies accommodated by the 


252 


system. Finally, we outline work now in progress to further generalize the 
Bugsystem to provide for the analysis of movement in three dimensions. 


DEFINING A BEHAVIORAL RESEARCH LANGUAGE 

The Behavioral Research Language, or BRL, is a high level operator based 
language that is tailored to the unique problems associated with the input, 
scaling, analysis and display of video images of moving objects. BRL is an 
interpretive language which runs as an application program on a good sized 
minicomputer and relies heavily upon user interaction with a storage graphics 
terminal to input, plot, edit and transform the data through image processing 
functions. Image processing generally culminates in the computation of paths 
or trajectories of the objects moving before the video camera. Sets of 
trajectories may be merged together and the data may be transformed to yield 
time series of behavioral variables (e.g., linear velocity, angular velocity, 
direction of travel, etc). These results may then be analyzed statistically, and 
the resultant data sets either listed numerically or plotted on the graphics 
terminal. Thus, the Bugsystem consists of two basic subsystems: (1) an unique 
image processing system for the frame-by-frame analysis of video data; and (2) 
a signal processing system for the statistical analysis of equispaced time series. 

The key element to understanding and using BRL lies in grasping the 
operator-operand-resultant nature of specifying functions or commands to the 
system. The general command syntax is as follows: 

*Operator/sw/sw Operand-name/sw/sw Resultant-name/sw/sw nl, . . . n5, 

Where “*” is the prompting character, “Operator” is one of the available 
functions (of which there are currently 88, with the list still growing), 
“Operand-name” is the name of the input (or operand) data set, and the 
numeric constants “nl” through “n5” are optional numeric constants which 
govern details of the function of certain operators. The “/sw” denote optional 
“switches” (the word is taken from minicomputer jargon) which are used as 
operator modifiers or to supply special information to the operator being used. 
All data sets are disk resident and are specified by a four-letter name that can 
be used to denote an experimental condition, a two-letter extention that 
specifies the type of data represented (e.g., “VI”-video, “PA”-path, 
“LV’Minear velocity, “CA”-catergorized, etc.), and a six letter front name that 
can be used to identify the species studies and/or the date of the experiment. 
The front name must be specified only when starting the system and remains 
unchanged unless it is explicitly modified by the LOAD operator. Operator 
names will henceforth be in bold upper case letters in the text. 


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For example, to plot all of the data in the video file called BUGS.VI, the 
command would appear as “*PLOT BUGS.VI”. To plot only the fust twenty 
frames of this file, one would enter “*PLOT BUGS,VI 1,20”. To calculate the 
linear velocities for the paths in the Fde BUGS.PA, the command line is 
“*LVEL BUGS.PA BUGS.LV”. Note that in this language the loops required 
to access all data elements within a data set are not explicitly stated. The 
operator automatically processes all of the elements of the operand data file 
unless directed to a particular subset (e.g., “*PLOT 1, 20”, as illustrated 
above). 

Three other aspects of BRL are worth including here. The first of these 
concerns the way in which data are represented within the Bugsystem: a data 
set consists of one or more vectors of variable length. While performing image 
processing operations, each vector represents one video frame; one element of 
such a vector represents a single point in two dimensional space. As the analysis 
of the data proceeds through successive application of operators to operand 
data sets, a single vector may represent an organism’s path (i.e., a time series of 
cartesian coordinates in two space as in the tile “BUGS.PA” illustrated above) 
or a real function defined over the length of such a path (e.g., the estimate of 
instantaneous linear velocity as previously illustrated by the file “BUGS.LV”). 
Finally, in the statistical analysis of such data a vector may correspond to a set 
of statistical parameters, a collection of “bins” or categories established for 
histograming, an estimate of an autocorrelation function, etc. 


The second aspect of the language to be considered here are the 
self-documenting aspects of BRL. No one can be expected to memorize all of 
the 88+ operator names, what they do in detail and the various switches and 
numeric constants which they expect. To help in this regard, the NAMES 
operator lists on the terminal the names of all the keyboard operators. 
Moreover, entering “*Operator/HELP” for any of the available operators will 
cause the system to print a full page of information describing what the 
operator does, the types of operands for which the operation is defined, what 
constants and switches are expected and an example of the operator’s use. 

The third aspect of BRL to be considered involves the construction of user 
programs. BRL was designed primarily to be an interactive language: the user 
normally enters commands at the terminal one at a time and thereby directs 
the analysis to its desired end. It is also possible to create a disk file of 
commands consisting of operators and operand specifications and to direct the 
system to execute this stored sequence of operations (a computer program 
written in BRL) via the USER operator. 


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THE IMPLEMENTATION OF BRL 


The implementation of BRL by means of the second generation Bugsystem 
differs significantly (both in hardware and in software) from that of the 
prototype system. This section outlines the main features of the new system, 
giving reasons for their importance. 

A New Bugwatcher 

The Bugwatcher hardware was redesigned to utilize medium scale 
integration (MSI) circuitry. Algorithmic state machine (ASM) charts were 
employed to formulate and to document the new design. Functionally, the 
new Bugwatcher is similar to its earlier counterpart: it extracts digital X-Y 
coordinate pairs representing points belonging to image outlines from the video 
raster scan and stuffs these coordinates into the computer’s direct memory 
access (DMA) channel. The computer program to input these data buffers them 
and writes them to disk, thereby allowing the collection of extremely long data 
records. Within such a record each frame of video data is represented as a 
vector. Each video vector sent by the Bugwatcher to the computer contains not 
only a variable length list of coordinate data, but also includes a leading header 
of fixed length. The elements of this header are referred to as “vector 
attributes” and are employed to encode information associated with each 
vector. Video data possess four 16-bit words of attribute information supplied 
by the Bugwatcher hardware: (1) a unique word consisting entirely of zeroes 
used by the software to delimit frame boundaries; (2) a descriptor word which 
contains an encoding of the frame rate at which the video data were digitized 
and the on-off status of tone stimulus markers; (3) a total frame counter that 
can be used to determine relative or absolute time intervals between data 
segments recorded at varying time intervals; and (4) auxiliary digital input 
which allows an encoding of an experimental variable (e.g., the direction of the 
source of stimulation) to be automatically associated with each video frame. 
Representation of stimulus conditions is discussed in more detail below (see 
“Coupling to Research Environments”). This division of vectors into attributes 
and data applies to all vectors manipulated by Bugsystem software. However 
the meaning of each attribute depends on the type of data; e.g., one of the 
attributes of a path (represented as a single vector) is the starting frame 
number. 

The Implementation Language—FORTRAN IV 

The prototype BRL system was implemented entirely in assembler language 
on an IBM 1800 computer and ran as a stand-alone system with no operating 
system support. This made it non-portable and difficult to maintain and 
expand. The new BRL system was to overcome these major shortcomings; 


255 


thus, we chose FORTRAN IV as the language with which to implement the 
Bugsystem. FORTRAN IV has become a standard language among 
minicomputers and has simplified the tasks ot maintaining Bugsysem software 
and training new programmers to implement new BRL operators. The 
importance of these aspects cannot be overestimated since, all in all, ten 
different programmers have added software to the system during its three years 
of development, each requiring instruction on the software conventions and 
use of system utility subrouting packages. But at least they knew the 
FORTRAN language. 


The software was first operational on a PDP 11/45 computer at 
Southeastern Massachusetts University under the DOS-9 operating system. It 
was then implemented on a Data General ECLIPSE S/200 under the RDOS 
operating system. Some assembler language subroutines had to be recoded, 
including the software drivers that handle the direct memory channel to the 
Bugwatcher. But the transition to the new computer went fairly smoothly. The 
PDP 11 system was maintained for development purposes after the ECLIPSE 
was sent to its Narragansett home at the EPA lab. Due to malfunctioning 
DOS-9 software, the PDP 11 operating system was changed to RT 11—a change 
that required as much or more development effort than changing computers! 

Virtual File Structure 

Certainly one of the major disadvantages to using minicomputers for large 
application software projects is the address space limitation imposed by its 
small word size. This system was no exception. To overcome this limitation so 
as to allow both programs and data to fit within allocated memory, both 
program structures and data structures were designed so that only pieces of 
either resided in memory at any given time. Manufacturers of minicomputers 
recognize this problem and provide software support for manipulating 
overlayed programs; i.e., programs consisting of parts which are swapped in and 
out of main memory. However, software support was not available for similarly 
overlaying data sets. The earlier prototype system did not allow data sets to be 
any larger than the memory buffer on the IBM 1800—a simple solution, but 
not acceptable in the newer system. Certain Bugsystem applications require the 
acquisition and analysis of behavioral records consisting of many frames of 
video data or the trajectories of hundreds of organisms. The system required 
the potential to manipulate data structures ten to hundreds of times the size of 
main memory available for data. 

One of the early software design goals was to simplify the process of adding 
new BRL operators. Each operator was to be implemented in FORTRAN as a 
program overlay. An operator would be required to access as many as three 


256 


simultaneous data sets (e.g., the arithmetic operators PLUS, SUBTRACT, 
MULTIPLY and DIVIDE each require two operands and generate one 
resultant) within a labeled common buffer of 8192 integer values (or 4096 
single precision values). Within the code that implements a new operator, files 
are accessed with a complete set of virtual file handlers coded in FORTRAN. 
These routines provide services for opening an existing file, creating a new file, 
reading a vector into the buffer from disk, writing a vector from the buffer to 
the disk and closing a file. To minimize disk access time and thereby insure 
optimum response time to the user, techniques employed in managing other 
virtual memory systems were adapted to the Bugsystem. All data sets, stored 
on disk as contiguous files, are accessed directly using multiple block transfers. 
If, for example, a command is issued from an applications program to read a 
given vector within a file, then the software first determines if the vector is 
resident within the buffer. If it is resident, then the routine immediately 
returns to the calling program providing the length of the vetor and a pointer 
into the buffer to the first element of the vector. If it is not resident, its logical 
address within the file is computed and it—and the vectors which succeed 
it—are read into the labeled common area. A similar algorithm is employed 
when writing a vector into a file; i.e., a disk write is not required unless the 
output buffer area is full. 

Funneling all file input/output through a common set of routines has 
significant advantages. The development of new BRL operators is simplified 
insofar as the underlying applications programs do not each separately (and 
redundantly) require the algorithms needed to manipulate large files. Another 
advantage lies in the increased portability of the software. Versions of 
FORTRAN supported by different machines and operating systems vary most 
markedly in their non-standardized methods of accessing files. Machine and 
operating system dependencies are thus isolated in a manageable number of 
software modules, yielding more portable and more easily maintainable 
software. 

COUPLING TO RESEARCH ENVIRONMENTS 
Images 

Primary input to the Bugsystem consists of digitized video data. If the video 
images are sufficiently “clean” (i.e., possess high contrast and lack structural 
complexity), input of data to the computer is accomplished automatically in 
real time. Video tapes are replayed into the Bugwatcher which compares the 
incoming video signal to a “video threshold” set by the user. Those points 
within the image where the video signal crosses the video threshold are 
displayed on a video monitor. The user adjusts the video threshold to make 
these points coincide with the outlines of moving organisms and selects a frame 


257 


rate (ranging from sixty frames/sec to one frame/min) appropriate for the 
relative speed of the organisms. When the BUGWATCHER INPUT operator is 
executed, the computer accepts digitized image information from the 
Bugwatcher at the selected frame rate. Each threshold point is represented in 
Cartesian coordinates with 8-bit resolution for each of two orthogonal 
components. A video frame is represented within the resultant data structure as 
a data vector with a variable number of such points as its data elements. An 
entire record (or “video file”) consists of a temporally ordered sequence of 
such vectors. 

The Bugsystem was originally developed for the investigation of the 
behavior of motile microorganisms (2). In this application, the organisms are 
viewed under dark-field illumination swimming within a well slide upon the 
stage of a compound microscope. However, automatic digitization of data is 
possible for any study of moving objects for which “clean” video records are 
available. One of us (Wilson) is currently using the second generation system to 
study the effects of plane polarized light upon the behavior of aquatic 
arthropods. The animals move freely within a cylindrical aquarium (diameter » 
20 cm) under bright-fleld illumination and are viewed using a macro lens 
attached to the television camera. Use of video tape as a storage medium allows 
experiments to be conducted in a laboratory remote from the site at which the 
data are analyzed. 

Occasionally video recordings are not “clean” enough to allow fully 
automated digitization (e.g., data collected in the field) or the digitized images 
are too crude to provide information about details of an organism’s anatomical 
structure (e.g., the orientation of its eyes). A technique has been developed to 
expedite manual analysis of such data. Using the PICK operators, the video 
tape is examined frame-by-frame. The user selects points upon the screen of a 
video monitor using a video cursor controlled by a JOYSTICK. A synthetic 
video signal representing a tiny bright dot is sent to the Bugwatcher which, in 
turn, sends digitized video information to the computer. Because averaging 
algorithms are employed, this method affords higher spatial resolution than 
does fully automated input: each coordinate in the resultant data structure 
may have 8-9 significant bits in comparison to the 7-8 bits of the normally 
digitized input data. 

Input of video data to the computer effects a mapping of the image as seen 
upon a television screen onto a two dimensional representational space. 
Distances within this space (“Bugspace”) are measured in arbitrary internal 
units (or “Bugwatcher units”) and, therefore, must be scaled. Using the LIVE 
INPUT operator, the Bugwatcher processed image of a ruler (or any object of 
known length)-recorded under the same conditions used in gathering 
behavioral data-is displayed on the screen of a CRT terminal and two points 


258 


are selected using the terminal cursors. Thus, the ratio between the distances 
separating the points in Bugspace and in physical space yields a factor or 
proportionality between Bugwatcher units and conventional units of spatial 
measurement (e.g., gm, mm, cm, etc.). 

Experimental Parameters and Experimental Variables 

Biological interpretation of behavioral data requires that the behavior of 
organisms be related to experimental conditions prevailing at the time of 
observation. Consequently, we have developed methods to associate 
experimental parameters and experimental variables with sets of data. An 
experimental parameter is a quantity characterizing a condition which is 
constant throughout any given record but may vary from record to record 
(e.g., temperature, concentration of a pollutant, etc.). Using the PARAMETER 
operator, such numerical constants may be inserted into the set of attributes 
belonging to each data vector. Parameters may be deleted, modified or listed. 
They may be used to organize graphical displays (e.g., average linear velocity as 
a function of temperature) or to modify the execution of certain operations. 

An experimental variable is a quantity whose value changes during a single 
record. Times at which simple step changes in stimulus conditions (e.g., 
switching a light on and off) occur may be indicated by the presence or 
absence of tones stored on the audio track of a video tape. The Bugwatcher 
possesses four tone generators to produce such temporal markers during the 
course of an experiment; it also possesses external connections which allow the 
simultaneous gating of laboratory apparatus. When the video tape is replayed 
into the Bugwatcher these tones are detected. As discussed above, the second 
attribute in each data vector sent to the computer contains four bits dedicated 
to representing the presence or absence of the four tones. 

We are presently developing a technique which provides for the 
representation of stimuli which vary continuously over time. The stimulus level 
will be encoded by means of frequency modulation (fm) on the audio track of 
the video tape. When the tape is replayed into the Bugwatcher the fm signal 
will be digitized and represented with 10-bit precision by the fourth attribute 
of each data vector sent to the computer. The APPEND operator will be 
employed to extract this information from each data vector, scale it and store 
as an equispaced time series. Again these data may be used to organize 
graphical displays or they may enter into computations involving time series of 
behavioral variables. One of us (Wilson) is preparing to employ this method to 
investigate behavior evoked by rotation of the plane of polarized light. The 
organisms swim beneath a polaroid filter whose angle is controlled by an analog 
servomechanism. The filter may be rotated so as to describe quick jumps, 
ramps, harmonics, etc. A signal directly proportional to the angle ol the filter 
will be encoded and analyzed. 


259 


PROCESSING AND DISPLAY 


Image Processing 

As discussed above, input of video data to the computer entails substantial 
preprocessing of pictorial information. A data vector within a resultant video 
file is a list of contemporaneous points; an organism’s outline is represented 
within this data structure as a localized set of points. The user can display such 
data graphically (using the PLOT operator) or alpha-numerically (using the 
LIST or EXAMINE operators). Video files may be edited both in time and in 
space. The EDIT operator allows one to save (or delete) temporally contiguous 
sets of data vectors. Thus, the user could EXAMINE the data to ascertain the 
frame at which the status of a tone had changed (indicating a change in 
stimulus conditions, e.g., switching on a blue light) and then EDIT the data to 
insure that this change occurs on frame number 100. The MASK operator 
allows one to save (or delete) points within rectangular or circular regions of 
the image plane. Thus, the user could MASK out all points within a video file 
which correspond to a particle of detritus within the experimental preparation. 
Finally, the user may APPEND additional information to a video file (text 
describing the conditions of the experiment, numerical constants, time series of 
tone states or time series of experimental variables). 

Analysis of video data by means of the Bugsystem proceeds by abstracting 
one (or more) points from each point set delineating an organism’s outline. In 
an investigation of translational movement this task is easily defined: unlike 
either rotational or flectional movement, quantitative description of the 
translational component of an individual organism’s behavior does not require 
detailed knowledge of the organism’s external anatomy. The body of the 
organism is represented by a single point, viz., its “center of mass”. 
Translational movement is defined as displacement of this point from one 
position in space to another. 

Mapping outlines into centrally located points is usually achieved by means 
of the CENTROID operator whose command syntax is exemplified by the 
entry 

*CENT BUGS.VI BUGS.CE N1, N2, N3. 

Each vector in the resultant file “BUGS.CE” corresponds to a vector in the 
operand file “BUGS.VI”. Each element of a resultant data vector is a 
“centroid”: a point in Bugspace whose X and Y coordinates are, respectively, 
the average X and Y coordinates of an “acceptable set” of points in the 
corresponding operand data vector. The numerical parameters “nl”, “n2” and 
“n3” are required to characterize an “acceptable set” of operand data points. 


260 


An initial member (having non-zero coordinates) of such a set is chosen from 
the operand data vector, the coordinates of this point are set equal to zero in 
the operand data and a mask (width = 2'nl, and height = 2*n2) is centered upon 
this point. The set is augmented if an operand point (having non-zero 
coordinates) falls within the mask. The mask is then centered upon the new 
point and the search continues. The search terminates when the set is deemed 
“acceptable” if it possesses at least n3 members. Ideally, this process yields one 
centroid corresponding to each outline—unless the outlines of two organisms 
are merged. The user can PLOT the centroids over the original data to confirm 
this correspondence and recompute the centroids using new parameters if the 
correspondence is not adequate. The structure of “centroid data” is quite 
similar to that of video data except that each coordinate of a centroid is 
represented with 15-bit (rather than 8-bit) precision in attempt to exploit the 
greater accuracy resulting from the averaging procedure. Centroid data is 
displayed and edited in the same fashion as video data. 

The PICK operator provides an alternative method to abstract points of 
interest from video data. The use of this operator for direct analysis of a 
videotape, thereby, bypassing the acquisition and processing of video files, is 
discussed above (see “COUPLING TO RESEARCH ENVIRONMENTS”). The 
user may also PICK points associated with each outline by means of the 
terminal cursors. The PICK operator is indispensable for investigations of 
rotational and flectional movement. For example, the user might elect to study 
the orientations of the longitudinal body axes of a group of organisms (i.e., 
simple rotation in a single plane). The longitudinal axis may be defined as a 
vector extending from the tail to the head of an organism. Thus, the user 
selects an ordered pair of points corresponding to each outline by entering 

*PICK/VI BUGS.VI BUGS.TH 

and using the cursors to specify first the “tail” and then the “head” associated 
with each outline. The resultant file “BUGS.TH” possesses the structure of 
centroid data with each “tail point” immediately followed by the correlated 
“head point”. These points are then segregated using the PLUCK operator. For 
example, the two commands 

*PLUC BUGS.TH BUGS.T 1,2 

*PLUC BUGS.TH BUTS.H 2, 2 

respectively produce the files “BUGS.T’-containing all first elements of the 
ordered pairs (viz., the “tail points)-and “BUGS.H”-containing all second 
elements of the ordered pairs (viz., the “head points). This type of analysis 
may be extended to encompass larger collections of points (or n-tuples), 
thereby providing for the study of flectional movement (e.g., the angles of 
propulsive appendages with respect to the longitudinal axis). 


261 


Regardless of the type of movement under investigation, the next stage of 
image processing entails the computation of paths or trajectories through 
Bugspace. A path or trajectory is a time-ordered set of points represented as a 
single data vector and characterized as follows: All points in a path are selected 
from an operand file consisting of centroids or having the structure of centroid 
data. The manner in which points are represented in a path is identical to the 
way they are represented in a frame of centroid data. Each path starts within a 
specific frame and its starting frame number is represented as the fourth 
attribute of the vector. Over the temporal interval during which a path is 
defined one (and only one) point is selected from each corresponding vector 
(or frame) of the operand file. Adjacent pairs of points within a path are 
selected on the basis of their spatial contiguity within adjacent frames. 

The search for paths is performed by the PATH operator whose command 
syntax is exemplified by the entry 

PATH BUGS.CE BUGS.PA nl, n2, n3, n4, n5. 

The numerical parameters “Nl” through “n5” control various details of the 
search, nl specifies the width (in Bugwatcher units) of a square mask used as a 
criterion of spatial contiguity. n2 is the maximum number of times to expand 
the mask if no contiguous point is located within the frame being searched. n3 
is the n4 minimum number of points to be accepted as a valid path. n4 is the 
minimum average displacement in Bugspace between consecutive frames for a 
set of points to be accepted as a valid path; this parameter may be used to 
“weed out” stationary artifacts (e.g., a particle of detritus). n5 is the maximum 
number of frames to “look ahead”; i.e., if no contiguous point is found in the 
current frame, then the next frame may be searched. If a point within the new 
frame qualifies, the missing point is computed by linear interpolation. 

Ideally, every path would correspond to the movement of a single point 
(associated with the outline of one individual organism) through Bugspace and 
each such movement would be represented by one path. In practice, the PATH 
operator may commit two types of errors. The operator may overlook certain 
segments of continuous movement. Such omissions may yield an abnormally 
short path, or they may result in a one-to-many (even a one-to-none) 
correspondence between real word trajectories and paths. Alternatively, the 
operator may confound certain segments of the continuous movements of two 
(or more) organisms. If there were but one organism in the field of view at any 
time, then errors of omission could be abolished by using a large mask (why 
not let it include all of Bugspace?) and allowing the program to look ahead 
several frames. But with several organisms represented within each frame there 
is clearly a tradeoff in choosing parameters so as to reduce the incidence of the 
two types of errors. The user may PLOT the paths over the centroids (or over 


262 


the original video data) to check their fit and then recompute the paths using 
new parameters. Often several iterations of this process are required to obtain 
an optimum combination of parameters. Since the parameters depend largely 
upon the magnification, the density of organisms and the way in which they 
move, the same “optimum set” of parameters (retained in memory for the 
user’s convenience) are generally used to compute paths for all replicates of an 
experiment. 

Proper selection of pathfinding parameters can significantly reduce the 
number of erroneous paths but cannot be relied upon to eliminate all errors. 
Consequently, we have developed programs which allow the user to interatively 
detect and correct mistakes within path files. Path editing programs (including 
CHOZ, EDIT, MERG and JOIN) allow the user to preform the following basic 
operations: (1) delete a path; (2) truncate a path; (3) cut a path into two 
smaller paths; and (4) join two paths (assuming they do not overlap in time). 

Once a valid collection of paths has been obtained, the user may proceed 
directly to the extraction of time series of behavioral variables (as discussed 
below) from the path files. Before doing so, however, there are several 
additional procedures which the user may choose to apply to the path data. 
Since the use of these procedures (and the order in which they are applied) is 
dependent upon the overall design of the experiment, we will first illustrate 
them by means of a specific example. 

Wilson video taped the behavior of Daphnia pulex (a small freshwater 
crustacean, commonly known as a “water flea”) in Talbot Waterman’s 
laboratory at Yale University. 12 animals were observed from below swimming 
against a brightly and uniformly illuminated background. A variable 
polarizer/depolarizer was interposed between the chamber containing the 
animals and the light source. The tape consisted of 23 separate video records. 
Before each recording the polarizing filter was rotated to a randomly chosen 
angle and, also in random sequence, the device was adjusted so as to polarize or 
depolarize the illumination. Each record began with the image of a strip of 
plastic attached to the filter in order to indicate the angle of the filter and the 
magnification of the image. The plastic strip was then removed and the animals 
were observed swimming under constant conditions. In all, 13 records were 
obtained under polarized light and 10 records were obtained under unpolarized 
light. Each recording lasted two minutes. 

The video tape was analyzed with the aid of the ECLIPSE at Narragansett, 
R.I. For each record the LIVE INPUT operator was used to determine the 
angle of the filter with respect to the Bugsystem reference frame (which is 
fixed with respect to the raster scan of the video signal). As discussed above, 
the appropriate spatial scale factor (approximately 0.40 mm/Bugwatcher unit) 


263 


was also computed. The BUGWATCHER INPUT operator was then used to 
generate one video file (480 frames of data at ten frames/sec. or 48 sec. of 
data) for each record on the tape. Wilson then MASKed the data, saving only 
those points within a centered circular region of Bugspace. The centroids and 
then the paths were separately computed for each masked video file using the 
same set of centroid and pathfinding parameters. Very few pathfinding errors 
resulted from these computations, but these were corrected by editing the 
separate path files. Wilson then employed the PARAMETER operator to 
associate the angle of the fdter with respect to the Bugsystem reference frame 
(an experimental parameter) with each path. This angle was the same for every 
path within a given path file. By repeated use of the MERGE operator, Wilson 
condensed the data to produce two exceedingly large files: one containing all 
paths observed under polarized light (154 paths, 13210 data points) and 
another containing all paths observed under unpolarized light (166 paths, 
13691 data points). These paths were still represented in Cartesian coordinates 
relative to the Bugsystem reference frame. However, orientation with respect 
to the plane of polarization should only be manifest with respect to the 
reference frame of the filter. Using the ROTATE operator, the two frames of 
reference were made to coincide: every path within each merged file was 
rotated in Bugspace (about an axis passing through the center of this space) 
through an angle obtained by negating the appropriate experimental parameter 
for each path. All resultant paths for Daphnia swimming under polarized light 
are PLOTed in Figure 17-1. Many of these paths may be seen to be aligned 
approximately orthogonal to the axis of the filter (i.e., perpendicular to the 
E-vector of the polarized light). 

The preceading analysis illustrates the use of three operators (vis., the 
PARAMETER, MERGE and ROTATE operators) to organize path data prior 
to the computation of behavioral variables. The ROTATE operator was 
implemented to expedite studies of animal orientation. Using this operator, all 
spatial data in a file may be rotated through a constant angle, each path may be 
rotated through a constant angle associated with that path or each point may 
be rotated through an angle associated with a corresponding moment in time. 
The last option enables the investigator to study orientation with respect to a 
moving stimulus (e.g., be MERGED) whenever the respective files may be 
taken to be replicates of the same experiment. Not only does this simplify the 
bookkeeping tasks associated with subsequent analysis, but, in addition, allows 
a set of similar data to be treated as a single sample by statistical operators. The 
fundamental advantage of an operator-based interactive system for the analysis 
of behavioral data is its flexibility: the operators which the user chooses to 

apply to the data-and the order in which they are applied—can be selected to 
correspond to the design of the original experiment. 


264 


FILE; PFIL SP 
VECTORS TOTAL 154 
DISPLAY RANGE ; 
FIRST : 1 

LAST: 154 










.. ' ■' ; ' - . 1 



Figure 17-1. Merged Path File of Daphnia Pulex Swimming with 
Respect to the Reference Frame of the Linear Polarizer. 


NOTE: The E-vector is horizontal to the field of view. The experiments and 
analyses which gave rise to these data are discussed in the text. 


Generating and Transforming Time Series Data 

Time series of X and Y coordinate values are generated from path data by 
my means of the SPLIT operator whose command syntax is illustrated by the 
entry 


*SPLI BUGS.PA BUGS.X BUGS.Y. 

Each data vector in the resultant files “BUGS.X” and “BUGS.Y” is, 
respectively, a time series of X and Y coordinate values. The resultant data sets 
are in one-to-one correspondence and each element is represented as a single 
precision floating point number. Like the paths from which they are derived, 
resultant time series may start and end at arbitrary points in time. Therefore, 
the number of series defined at any given moment is also arbitrary. Other 
computer systems have been developed for the analysis of equispaced time 
series (7). After image processing has been completed, the operators available 
on the Bugsystem are unique only insofar they possess the sophistication 
required to manage large collections of arbitrarily derived series (e.g., Figure 
17-2). 


265 





Figure 17-2. Linear Velocity as a Function of Time for 128 Paths 
of the Fairy Shrimp Eubranchipus Vernalis in Polarized Light. 


In principle, all behavioral variables which may be investigated using the 
Bugsystem can be generated by simple arithmetic transformation of series of X 
and Y coordinates. Greaves (4) has discussed the computation of certain 
behavioral variables (viz., linear velocity, net to gross displacement ratio, 
direction of travel and angular velocity) using arithematic operators 
implemented within the prototype system. Such operators transform every 
element of every data vector within a file, treating each vector as a separate 
unit of data. The present Bugsystem is also provided with a wide selection of 
simple arithematic operators. However, we have condensed the computation of 
certain frequently calculated behavioral variables into single operators which 
require path data as input. As an example, let us consider the RATE OF 
CHANGE OF DIRECTION operator whose command syntax is illustrated by 
the entry 


*RCDI BUGS.PA GUBS.RD nl. 

Every element in the resultant file “BUGS.RD” is the unsigned rate at which 
the corresponding path changed its direction of travel at a given moment. RCD 
is the absolute value of angular velocity expressed in degrees per second (The 
parameter “nl” is the frame rate, required to convert from degrees per frame.) 
The importance of this variable in ascertaining mechanisms responsible for 


266 






certain animal aggregations has been widely discussed (3). RCD may also be 
computed by a sequence of simple arithematic operations as follows: 

(a) *SPLIT BUGS.PA BUGS.X BUGS.Y 

(b) *CDIF BUGS.X BUGS.DX 

(c) *CDIF BUGS.Y BUGS.DY 

(d) UCTAN BUGS.DX BUGS.DY BUGS.DI 

(e) *CDIF BUGS.DI BUGS.AC 

(0 *MULT/CO BUGS.AC BUGS.AV nl 

(g) *ABSV BUGS.AV BUGS.RD 

Where “CDIF” is the CENTRAL DIFFERENCE operator (a discrete 
approximation to the differential operator), “CTAN” is the CONTINUOUS 
ARCTANGENT operator, “MULT/CO” denotes multiplication by a constant 
and “ABSV” is the ABSOLUTE VALUE operator. The resultant “BUGS.DI” 
of step (d) is the direction of travel measured in degrees with respect to the 
Bugsystem reference frame; it could have been generated from the original path 
data using the DIRECTION OF TRAVEL operator. The resultant “BUGS.AV” 
of step (e) contains angular velocities (measured in degrees per second); this file 
could have been produced using the ANGULAR VELOCITY operator. Other 
operators have been developed to evaluate LINEAR VELOCITY and NET TO 
GROSS DISPLACEMENT RATIO functions defined upon path data. 

Simple Statistical Processing 

For the purpose of statistical analysis two different types of data 
structure—representing two levels in a structural hierarchy-may be 
distinguished as “samples”: vectors and files. Many statistical operators 
recognize this distinction. For example the STATISTICAL PARAMETER 
operator estimates parameters such as the mean, variance, standard deviation, 
skewness, kurtosis, etc. The command 

*STAT/VE BUGS.LV BUGS.ST 

produces the resultant file “BUGS.ST’, containing one data vector (i.e., a list 
of statistical parameters) for each data vector in the operand, whereas the 
command 


*STAT/FI BUGS.LV BUGS.ST 

produces only one resultant data vector characterizing the entire file. In either 
case, the user can LIST the resultant parameters. Similarly, the SLOT operator 
provides for estimation of density and distribution functions via histograms 
both for individual vectors and entire files. These data may be displayed 
graphically (Figure 16-3) or LISTed on the terminal or the line printer. 

267 



Figure 17-3. Linear Velocity Histograms for E. Vernalis in 
Polarized Light (the Data Displayed in Figure 17-2) 
and in Unpolarized Light. 


NOTE: Dashed lines denote the estimated mean of each distribution. 


We chose to store statistical parameters within data vectors, rather than 
merely computing and displaying them, in order to allow them to enter into 
subsequent calculations. For example, assume the user had computed statistical 
parameters for each data vector in a file of instantaneous linear velocities (e.g., 
the “STAT/VE” example given above); each estimate of the mean is thereby an 
average for each path. The user may then investigate the distribution of these 
path averages. The estimated means are first isolated using the STRIP operator: 

*STRI/MN BUGS.ST BUGS.MN. 

The resultant file “BUGS.MN” contains a single data vector; each element of 
this vector is a mean (“/MN”) stripped from one operand data vector. The user 
may wish to compute statistical parameters for the new set of data, or explore 
its frequency distribution via histograms (Figure 16-4). The user may also 
MERGE such files so that each data vector corresponds to a single 
experimental condition. The vectors may then be compared with one another 
(e.g., one-way analysis of variance, chi-square tests, etc.). 


268 






Figure 17-4. Average Linear Velocity Histograms for E. Vernalis in 
Polarized Light and in Unpolarized Light 


NOTE: Dashed lines denote the estimated mean of each distribution. 


We have made special provision within the Bugsystem for the manipulation 
and statistical analysis of angular data. For example, the command 

*STAT/ AN/FI BUGS.DI BUGS.ST 

produces a single data vector whose elements are statistical parameters 
appropriate for circular distributions. These include the length and direction of 
the mean vector (and related measures) as discussed by Batschelet (1). 
Moreover, because the Bugsystem is well suited for investigations of animal 
orientation (and because this is a major interest of one of its codevelopers) we 
have implemented an extensive polar graphics package within the Bugsystem. 
Figure 17-5 illustrates the use of polar wedge histograms to represent angular 
density functions. 

More Advanced Statistical Operations 

These fall into two major categories: operators directed toward the analysis 
of time dependence of behavioral variables and operators used to explore 
mutual relationships between variables (e.g., Figure 17-6). In the First category, 


269 




Figure 17-5. Polar Wedge Histograms of Instantaneous Direction of 
Travel with Respect to the Filter Reference Frame for D. Pulex. 


NOTE: (A) in polarized light (evaluated for the path plotted in Figure 17-1) 
and (B) in unpolarized light. The percent of sample within each angular 
category is indicated on the radius. The dashed circles denote expectation for a 
circular uniform distribution. 







Figure 17-6. Dependence of RCD (Rate of Change of Direction) 
Upon Direction of Travel with Respect to the Axis of the 

Polarizer for D. Pulex. 

NOTE: (A) in polarized light and (B) in unpolarized light. Data was processed 
by partitioning RCD into disjoint subsets on the basis of the correlated 
direction of travel. The estimated mean (± the standard error of the mean) for 
each subset is indicated in the figures. 


271 





the Bugsystem includes several ensemble operators which compute a statistic 
(e.g., an estimate of the mean or mean vector or even a histogram) for every 
frame defined within the operand data. The Bugsystem also includes serial 
correlation operators (viz., AUTOCORRELATION and CROSS 
CORRELATION). Programs providing for analysis in the frequency domain, 
sinusoidal, regression and polynomial regression are currently under 
development. 

REFERENCES 

1. Batschelet, E. 1965. Statistical Methods for the Analysis of Problems in 
Animal Orientation and Certain Biological Rhythms. AIBS Monograph. 
Washington, D.C. pp. 1-57. 

2. Davenport, D., G.J. Culler, J.O.B. Greaves, R.B. Forward and W.G. Hand. 
1970. The Investigation of the Behavior of Microorganisms by 
Computerized Television. IEEE Trans. BME 17: 230-237. 

3. Fraenkel, G.S. and D.L. Gunn. 1961. The Orientation of Animals. Dover. 
New York, pp. 1-367. 

4. Greaves, J.O.B. 1971. An On-Line Television Computer System for the 
Study of the Behavior of Microorganisms. Ph.D. Dissertation. Dept. Elec. 
Eng., Univ. of California, Santa Barbara. 

5. Greaves, J.O.B. 1975. The Bugsystem: The Software Structure for the 
Reduction of Quantized Video Data of Moving Organisms. IEEE Proc. 63: 
1415-1425. 

6. Hand, W.G. and J.A. Schmidt. 1975. Phototactic Orientation by the Marine 
Dinoflagellate Gyrodinium dorsum Kofoid. II. Flagellar Activity and 
Overall Response Mechanism, J. Protozoel. 22: 494-498. 

7. Martin, W. and K. Brinkman. 1976. A Computer Program System for the 
Analysis of Equispaced Time Series. J. Interdiscipl. Cycle Res. 7: 251-258. 

8. Wilson, R.S. 1976. Light Elicited Behavior of the Marine Dinoflagellate 
Ceratium dens. Ph. D. Dissertation. Dept. Biol. Sci., Univ. of California, 
Santa Barbara. 


272 


THE EFFECTS OF TEMPERATURE, LIGHT 
AND EXPOSURE TO SUBLETHAL LEVELS 
OF COPPER ON THE SWIMMING BEHAVIOR 

OF BARNACLE NAUPLII 

William Lang 
Sarah Lawrence 
Don C. Miller 

U.S. Environmental Research Laboratory 
Narragansett, Rhode Island 02882 

ABSTRACT 

The “Bugsystem”, a computer-television system to accurately track and 
analyze swimming patterns of aquatic organisms has been developed; video 
images of test animals are converted to time sequence X-Y coordinates to allow 
rapid computer analysis of linear or angular velocity, rate of change of 
direction, direction of travel and other parameters. Initial experiments using 
barnacle nauplii (Balanus amphitrite, B. improvisus, B. venustus, Chthamalus 
fragilis) indicate larval swimming speeds are affected by temperature and light 
regime. Response to temperature appears to be function of species tested and, 
perhaps, geographic location of adult population. Changes in linear velocity 
induced by acute light intesity variation are of short duration. Mean linear 
velocities of nauplii are altered by 24 hour exposure to copper as low as 20 
ppb. Linear velocities of exposed populations increase relative to controls at 
low copper levels, and then decrease as lethal levels are approached. Copper 
will also alter the swimming pattern of exposed larvae. 

INTRODUCTION 

In view of concern that bioassays directed solely toward determining lethal 
concentrations of pollutants may not accurately reflect levels doing harm to 
the environment, attention has been directed toward sublethal effects of 
pollutants- “effects which do not immediately, or directly, lead to death, but 
which nevertheless cause disturbances which may be of ecological significance 
(1).” Existing studies using pathological, physiological, and behavioral 
parameters indicate approximate thresholds for sublethal responses are often 
10-20 percent of LC50 levels or less (7, 22). It is generally recognized that 
behavioral responses of marine animals are often highly sensitive to stress (18) 
and that juvenile or larval stages of many marine organisms represent that part 
of the life-cycle most susceptible to stress (4, 8, 16). Logically, larval 


273 


behavioral responses to pollutants would represent a potentially significant 
field of study; however, devising a means to easily record and rapidly quantify 
swimming and other responses of small larvae has limited efforts in this 
direction (8, 12, 17, 24). 

Development of the Bugsystem at this laboratory has provided the 
technology to rapidly analyze the swimming patterns of large sample numbers 
of organisms of a wide size range (Wilson & Greaves, this volume, report 17). 
With this potential we are presently investigating the use of behavioral 
bioassays for marine larvae. The following results represent initial studies using 
larvae of common barnacle species. 

EXPERIMENTAL 

The spontaneous locomotory activity for second stage nauplii of four 
barnacle species ( Balanus amphitrite amphitrite, B. improvisus, B. venustus, 
Chthamalus fragilis ) was investigated. Of primary concern in this initial study 
was the mean linear velocity (MLV) of sample groups and changes in MLV 
induced by water temperature, light regime, and 24 hour exposure to sublethal 
copper levels. 

Source of Larvae 

Second stage Balanus nauplii used in all experiments were released from 
adult barnacles maintained at 20 ± 2°C, constant illumination in 30-32°/oo, 1 
p fdtered seawater from Narragansett Bay. Balanus amphitrite and B. 
improvisus were initially collected near Georgetown, South Carolina, and 
maintained under the above laboratory conditions 2-12 weeks prior to larval 
release. B. venustus adults were collected in Narragansett Bay; adults released 
larvae within two weeks after capture. Chthamalus fragilis nauplii, also from 
Narragansett Bay, were obtained from ripe egg masses incubated for 24 hours 
at 20°C. In some copper experiments, stage II nauplii were reared to later 
stages on a mixed algal diet of Tetraselmis suecia and Thalassiosira pseudonana . 
Methods of maintaining laboratory populations of barnacles and rearing of 
nauplii are further described by Lang (13, 14). 

Video Recording 

To obtain video tapes of swimming patterns for Bugwatcher analyses, ca.30 
nauplii were placed in 25 ml beakers with a water depth of about 10 mm. The 
beaker with nauplii was put within a cylindrical flat black metal lenshood (light 
shield) attached to a 72 mm diameter #25 deep red glass filter (Figure 18-1). 
This complex was centered upon a Wild M-5 dark Field illumination stage Fitted 
with an “800 nm” interference filter and 22 mm diameter diaphram (Figure 


274 


A 


B 


C 

\_ . / 

t _. r p 


H 



Figure 18-1. Diagram of Video Recording Equipment. 

NOTE: A) TV camera, B) Photo tube, C) Wild M-5 microscope, D) wide-angle 
attachment, E) metal light shield with deep red glass bottom, F) container with 
test organisms, G) 800 nm interference filter, H) metal diaphram, I) clear glass, 
J) dark field stage with halogen light. Components E-H are shown separated 
from each other for graphic clarity. 


18-1). Video images were obtained using a Cohu 4400 television camera 
attached to the M-5 microscope body. The field of view recorded was 10x10 
mm. Optimum image contrast was obtained using a halogen light source with 
dark field optics at 8.5 volts (Figure 18-1). 

Spectroradiometer (ISCO-SR) readings indicate light transmitted through 
the stage filters was as low as 680 nm. Peak transmission occurred between 
810-840 nm. With the exception of light experiments, nauplii were moved 
directly from constant light temperature boxes to the microscope stage. Room 
lights were extinguished and, following a two-minute acclimation period, larvae 
were taped for 3-5 minute intervals using filtered substage illumination. 
Swimming parameters reported were determined by analysis of 30-60 second 
portions of these tapes; results are pooled from replicate samples. 


275 






















Response to Light 

Nauplii used in all experiments were light adapted as fluorescent bulbs in 
the temperature boxes were on continuously. When transferred to the darkfield 
stage, larvae tended to disperse to the beaker walls with ceiling lights on. With 
these lights extinguished, nauplii tended to swim away from the beaker walls. 
Direction of travel upon entering or leaving the camera field exhibited no 
particular orientation. Ongoing studies indicate dark adapted B. amphitrite 
nauplii will exhibit a weak photonegative response to substage light over a 
five-minute period (Forward & Lang, personal observation). 

Balanus spp. stage II nauplii exhibit similar response to sudden changes in 
light intensity. When overhead white room lights are turned on, Balanus nauplii 
will approximately double linear velocities, then within 4-6 seconds return to 
initial swimming speeds (Figure 18-2). Turning overhead lights off has 
essentially the opposite effect; nauplii will cease locomotion for about five 
seconds and then return to initial swimming speeds (Figure 18-3). 



Figure 18-2. Example of running average linear velocity 
(mm/sec) for sample of ten stage II Balanus venustus 
nauplii exposed to sudden light increase. 


NOTE: Dashed line indicates time at which overhead white light stimulus was 
applied. Filtered (820 nm peak transmission) substage light was present 
throughout experiment for recording purposes. 


276 















Figure 18-3. Example of reaction of a single stage 11 Balanus 
venustus nauplius to sudden light decrease. 


NOTE: Dashed line indicates time at which overhead white light was extin¬ 
guished. Filtered (830 nm peak transmission) substage light was present 
throughout experiment for recording purposes. 


Chthamalus fragilis exposed to similar light changes exhibited little response 
in terms of MLV. The distinctive response seen with Balanus nauplii was clearly 
absent. 

Response to Temperature 

Newly hatched nauplii from the same brood (incubated at 20°C) were 
subdivided and placed into various temperature boxes for 24 hours to test the 
effects of temperature on swimming velocity (Table 18-1). The metal 
lightshield, glass filters, and beaker with larvae were equilibrated to the test 
temperature, then transferred immediately to the microscope stage for brief 
taping. Readings with a temperature probe indicated a maximum 2°C shift 
toward ambient occurred during taping. 

Even with the potential of a 2°C deviation in test temperatures, certain 
geographical distinctions are suggested between swimming velocity and 
temperature (Table 18-1). Data from Balanus amphitrite and B. improvisus 
nauplii from South Carolina adults suggest a direct relationship of increased 
swimming velocity with increased temperature. In contrast, B. improvisus 


277 












Table 18-1. Mean Linear Velocities (MLV) of Stage II 
Barnacle Nauplii Exposed to Different Temperatures 

for 24 Hours. 


Species 

Temp. 

(°C) 

N 

MDP 

(Sec) 

MLV ± sd 
(mm/sec) 

T test 

BA 

28 

12 

8.5 

0.81 ± 0.23 

NS 

BA 

20 

24 

9.0 

0.71 ±0.34 


BA 

15 

22 

7.1 

0.58 ± 0.20 

P=.20 

BIS 

25 

26 

7.4 

1.18 ± 0.56 

NS 

BIS 

20 

26 

6.0 

1.09 ± 0.43 


BIS 

15 

25 

8.0 

0.72 ±0.43 

P=.05 

BIN 

26 

13 

7.3 

1.56 ±0.62 

P=. 10 

BIN 

22 

17 

9.4 

2.43 ± 0.64 


BIN 

10 

14 

8.0 

1.25 ± 0.64 

P=.05 

CF 

25 

26 

7.2 

1.46 ±0.48 

P=.05 

CF 

20 

40 

6.4 

1.92 ±0.67 


CF 

15 

40 

6.0 

1.89 ±0.62 

NS 


nauplii from Rhode Island adults exhibited a decreased velocity above 22°C. A 
comparable reduction in velocity with Rhode Island nauplii also occurred with 
Chthamalus fragilis above 20°C. Yet overall, naupliar swimming speeds appear 
to be greater in the Rhode Island animals when measured within this 
temperature range. 


Brood Variability 

Linear swimming velocity in nauplii obtained from different broods of 
South Carolina adults was assessed under similar temperature, salinity, and 
light regimes to evaluate brood variability. Linear velocities were found to be 
similar among six broods (Table 18-2). Examples of MLV distributions within 
test groups of B. amphitrite , C. fragilis , and B. improvisus are shown in Figure 
18-4. Control groups usually have linear velocity distributions which 
approximate normal or are skewed to the left. 


278 





Table 18-2. Mean linear velocites (MLV) of various hatches of 
stage II Balanus amphitrite nauplii at 20°C. 


Hatch 

No. 

N 

MDP 

(sec) 

MLV ± sd 
(mm/sec) 

3 

40 

7.0 

0.75 ± 0.34 

8 

57 

5.3 

0.72 ±0.20 

9 

14 

6.1 

0.65 ±0.43 

14 

24 

9.0 

0.71 ±0.23 

24 

23 

7.4 

0.72 ±0.27 

25 

20 

7.0 

0.85 ±0.33 


NOTE: N = number of paths analyzed, MDP = mean duration of paths 
analyzed. 1 1 ' * ' ’ 1 1 f 



% 



30 .. 

20 -• 

10 - 

0 
30 

20 -■ 

10 .. 

0 

30 .. 

20 - 

10 



—I- >~ 

1 

M M/SEC 


D 


Figure 18-4. Examples of mean linear velocity (mm/sec) dis¬ 
tribution within five test groups of stage II barnacle nauplii: 


NOTE: A) Balanus improvisus from Rhode Island at 20°C, B) Chthamalus 
fragi/is at 20°C, C) Balanus amphitrite control, D) B. amphitrite with 24 hr. 
exposure to 20 ppb Cu, E) B. amphitrite with 24 hr. exposure to 350 ppb Cu. 
N = number of paths per group. 


279 












































































Response to Copper 


Having characterized linear swimming velocities for B. amphitrite nauplii 
under defined conditions, the effects of sublethal levels of copper on swimming 
speeds were investigated. A primary stock solution of 10,000 ppm Cu in 
dilute nitric acid was adjusted to 1-4 ppm secondary stock solutions using 
deionized water. Final test solutions were obtained by serial dilutions with 1 p 
filtered natural seawater at 32-34°/oo. Total copper was determined by heated 
graphite absorption on a HGA-2100 coupled to a Perkin-Elmer 360 atomic 
absorption spectrophotometer. 

Newly hatched B. amphitrite nauplii were exposed to various copper levels 
for 24 hours at 20°C. Replicate samples of nauplii for each exposure level were 
then video taped in a darkened room using the described darkfield 
illumination. Total mortality at 24 hours post-exposure was determined. 
Larvae were not fed during the experiment. Analyses indicated a 3-15 percent 
decrease in total copper occurred during a 24-hour static exposure. In the first 
test, nauplii were exposed to levels ranging from control (3 ppb) to 
approximately 50 ppb Cu (Table 18-3). No increased mortality was observed; 
however, MLV of nauplii exposed to 10 through 47 ppb Cu nearly doubled 
relative to control values. Close agreement was observed in replicate samples. 

In a second test, three higher copper levels were added: 120, 185, 350 ppb. 
After the 24 hour Cu exposure period, nauplii were transferred to clean filtered 
seawater with the mixed algal diet. Significant mortality at 24 hour 
post-expossure occurred only at 350 ppb Cu. However, a delay in molting was 


Table 18-3. Mean Linear Velocities (MLV) of 
Balanus Amphitrite Stage II Nauplii Exposed to Sublethal 
Copper Levels for 24 Hours at 20°C 


Copper 

(ppb) 

No. 

Animals 

Mean Path 
Duration (sec) 

MLV ± sd Mortality 24 Hr. 

(mm/sec) Post Exposure 

3 

16 

6.4 

0.71 ±0.27 

3% 

3 

14 

6.2 

0.65 ± 0.44 

5% 

10 

12 

5.0 

1.24 ±0.48 

4% 

12 

10 

7.3 

1.18 ± 0.51 

2% 

14 

21 

7.6 

1.09 ± 0.54 

2% 

14 

6 

7.3 

1.06 ±0.76 

4% 

30 

10 

7.8 

1.07 ±0.54 

2% 

47 

15 

6.4 

1.25 ± 0.47 

5% 

47 

11 

7.0 

1.16 ±0.48 

5% 


280 





seen at 48 hours post-exposure; only 35 percent of nauplii exposed to 185 ppb 
Cu had molted to Stage III, as opposed to nearly 70 percent at lower copper 
levels. (Figure 18-5.) Analysis of variance for naupliar MLV indicated a 
significant (p=0.01) difference between control (2 ppb), intermediate (18-186 
ppb), and highest (350 ppb) copper levels. Swimming speeds significantly 
increased at sublethal copper levels but rapidly declined at or near the lethal 
level (Figure 18-6.) The shift in distribution of linear velocities for test groups 
at these three exposure levels is clearly illustrated by frequency histograms 
(Figure 18-4). 

In the third experiment, B. improvisus nauplii (from South Carolina) were 
exposed to Cu levels ranging from control (3 ppb) to 190 ppb for 24 hours at 
25°C. Following video taping at 24 hours, nauplii were transferred to filtered 
seawater with algal diet and reared at 25°C. At 24 hours post-exposure 
mortality at 190 ppb was 100 percent; at 150 ppb, 20 percent. Rearing to 
cyprid stage indicated no significant mortality differences between controls 
and nauplii exposed up to 98 ppb Cu. However, development time appeared 
delayed by Cu exposure as low as 50 ppb (Table 18-4). 



Figure 18-5. Mortality 24 Hours Post Exposure and Percent 
Larvae Molting to Stage III, 48 Hours Post Exposure, for 
Balanus amphitrite Stage II Nauplii Exposed to Various Copper 
Concentrations for 24 Hours at 20°C. 


281 






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Figure 18-6. Mean linear velocity (mm/sec) of stage II barnacle 
nauplii following 24 hour exposure to different copper 

concentrations. 




























Table 18-4. Percent mortality and development time to cyprid 
of Balanus improvisus larvae following 24 hour exposure of 
stage II nauplii to different copper concentrations at 26°C. 


Cu 

(ppb) 

N 

Mortality 

(%) 

Days to Cyprid 
Range 

Mean 

3 

75 

19 

7-15 

10.2 

25 

81 

25 

7-15 

10.7 

47 

59 

19 

8-16 

11.7 

77 

64 

19 

9-16 

12.6 

98 

77 

25 

9-14 

11.6 

150 

76 

61 

8-15 

10.3 

190 

70 

100 

— 

— 


NOTE: During the post-exposure period, larvae were reared on a mixed algal 
diet of Tetrase/mis suecia and Tha/assiosira pseudonana at 26 ± 1°C. 


Swimming speeds of the above nauplii were significantly (p=0.01) increased 
at 25 and 50 ppb Cu relative to controls, nearly indentical at 98 ppb Cu, and 
significantly decreased at 150 ppb Cu (Figure 18-6). Although MLV of control 
and 98 ppb Cu populations were equal, the rate of change of direction (RCDI 
— absolute value of angular velocities in degrees/second) was substantially 
different (Table 18-5). These data show that the nauplii not only changed 
swimming speeds but also tended to change swimming patterns with increasing 
concentrations of Cu. Examples of path outlines and their computer assigned 
RCDI values are shown in Figure 18-7. 


Table 18-5. Rate of Change of Direction (RCDI) and 
Mean Linear Velocities of Balanus Improvisus Stage II 
Nauplii after 24 Hour Exposure to Indicated 
Copper Levels at 26°C 


Cu 

(ppb) 

N 

Linear Velocity 
(mm/sec) 

RCDI 
(deg./sec) 

3 

40 

0.82 

130 

24 

40 

1.43 

140 

57 

40 

1.23 

151 

98 

40 

0.83 

166 

151 

29 

0.49 

188 


283 











Figure 18-7. Examples of computer tracked paths for stage II 
Balanus improvisus nauplii and assigned rate of change of 
direction values (degrees/sec.): A) 114, B) 53, C) 152, D) 373. 


NOTE: Paths A, B are typical of control conditions; path C occurs more fre¬ 
quently with copper present; path D was observed only above 50 ppb copper. 


DISCUSSION 

Initial results have demonstrated possible use of invertebrate larval 
swimming behavior as a sublethal response index. It has also been shown that 
for this index to be reliable, the effects of basic experimental variables such as 
temperature and light regime on the swimming response of test organisms 
should be understood. 

Although previous studies on cirriped and brachyuran larvae (2, 11, 25) 
indicate no phototactic response is evident above 650 nm, cirriped nauplii 
appeared to exhibit a weak response to the present substage light. Spectral 
sensitivity of the species tested appears to extend further into the red than 
previously reported. 

Balanus venustus and B. amphitrite nauplii exhibited two responses to 
sudden changes in light. The cessation of swimming by nauplii following a 
sudden light decrease is similar to the “sinking response” described for 


284 





brachyuran larvae (10). The increase in swimming velocity of larvae following 
light increase cannot be fully described without consideration of possible 
directional response. Of practical relevance to our video taping and analysis 
procedures is that both responses are of brief duration, limited to the first 
three to five seconds of an acute illumination change, and can be detected from 
calculations of the MLV. The significance of these responses are not yet 
understood. Similar behavioral characteristics were not observed in stage II 
nauplii of Chthamalus and are also reported lacking in Balanus balanoides (6). 


Temperature is known to directly affect swimming rate of invertebrate 
larvae (15, 23). All temperatures tested here on second stage nauplii were 
within ranges allowing complete development of the barnacles (14). It is clear 
for all species tested that small temperature shifts can alter swimming speeds. 
The basic influence of temperature observed on larval swimming rates is 
probably primarily a function of species and thermal history, yet initial results 
with these barnacle nauplii suggest other factors may prove significant. For 
example, Balanus improvisus collected from Rhode Island and South Carolina 
and maintained at identical laboratory conditions for over one month, released 
larvae having apparently different swimming rates relative to temperature. 
Maximum MLV occurred at 25°C for South Carolina larvae and at 22°C for 
Rhode Island larvae. Similar differences in response were observed in B. 
amphitrite from South Carolina and C. fragilis from Rhode Island. Replicate 
tests with different hatches are needed to confirm whether geographical 
variations persist. 

To determine whether swimming patterns of barnacle nauplii are altered by 
toxic substances, stage II nauplii were exposed to different copper 
concentrations. Our exposure time to copper was limited to 24 hours. No algae 
food was added during this period to preclude complexing of the metal by the 
algae. Deprivation of food for 24 hours is not deleterious to the larvae. A 24 
hour LC50 of between 200-350 ppb Cu for B. amphitrite nauplii at 20°C and 
between 150-200 ppb Cu for B. improvisus nauplii at 26°C was observed. 
Similar toxic concentrations have been reported for Balanus crenatus nauplii 
(19) and Balanus eburneus nauplii (5). Cyprid larvae or adults were more 
resistant to copper in both these studies. 

Differences in LC50’s observed for B. amphitrite and B. improvisus nauplii 
may be related to temperature. Higher temperatures can increase copper 
toxicity (3) or at least give this appearance in short-term experiments (21). 
Weiss (26), however, found B. amphitrite to be more tolerant of Cu than B. 
improvisus at settlement. In either case, toxic etlects of Cu are often 
cumulative (3); both LC50 levels and sublethal effects probably occur at lower 
concentrations with increased exposure times. 


285 




For 24 hour exposures, concentrations of Cu below 100 ppb were clearly 
sublethal to the nauplii tested. Delay in development of B. improvisus nauplii 
occurred at 50 ppb Cu and changes in swimming behavior were evident at 
15-25 ppb Cu. At the lowest Cu test levels, responses were restricted to 
increased MLV, but at higher sublethal concentrations MLV was depressed and 
swimming patterns became atypical. A stimulatory effect of very low levels of 
copper on swimming activity has also been reported for brook trout (9.5 ppb 
Cu) (9) and with sea urchin sperm (<20 ppb Cu) (28). 

Forward and Costlow (12) also observed increased swimming activity of 
crab larvae exposed to 0.1 ppm of an insect juvenile hormone mimic, although 
larval development was not perceptively affected until 1.0 ppm was reached. 
On the other hand, sublethal concentrations of mercury and oil are reported to 
depress activity of marine crustaceans at nearly all levels tested (8, 18). 
Stebbing (23) suggests that apparent stimulatory effects of heavy metal ions on 
growth in marine hydriods and other groups are often only temporary and may 
represent a normal response to stressors. 

Observations on swimming of B. improvisus nauplii indicate that not only 
the linear velocity, but also the pattern is altered by Cu. Nauplii swimming in 
convoluted paths (Figure 18-7) tends to increase in number in the presence of 
copper above control levels. As copper concentrations exceed 50 ppb, paths 
with a distinct “wobble” became evident (Figure 18-7). This latter pattern is 
possibly a consequence of impaired or abnormal beating of appendages. This 
would lead to reduced feeding abilities, as feeding in cirriped larvae is a direct 
function of appendage movement. The increased development time to cyprid 
observed at higher sublethal copper concentrations may be the result of 
difficulties in feeding. 


The present study has consistently observed altered swimming behavior of 
cirriped larvae at Cu concentrations far below 24 hour toxic levels. Basic 
changes in swimming speed per se may prove useful indicators of pollution 
stress, but also of great interest are additional effects on larval motile responses 
to environmental stimuli or cues (light, chemical, gravity, etc.). The latter may 
prove more meaningful in predicting safe levels of pollutants. If short-term 
behavioral reaction can be satisfactorly correlated with long-term detrimental 
effects, the potential exists for rapid screening of toxic levels using this motile 
behavioral qualification technique. Further studies relating observed behavioral 
responses to other physiological parameters, and ultimately larval success, are 
planned. 


286 



ACKNOWLEDGEMENTS 


We wish to acknowledge the assistance of Gerald Hoffman and Raymond 
Zanni for providing copper analysis; and Richard Steele and Leslie Mills for 
culturing algae used to rear larvae. 

REFERENCES 

1. Anderson, J.M. 1971. II. Sublethal Effects and Changes in Ecosystems, 
Assessment of the Effects of Pollutants on Physiology and Behaviour. Proc. 
R. Soc. London B. 177: 307-320. 

2. Barnes, H. and W. Klepal. 1972. Phototaxis in Stage I Nauplius Larvae of 
Two Cirripedes. J. Exp. Mar. Biol. Ecol. 10: 267-273. 

3. Bryan, G.W. 1976. Some Aspects of Heavy Metal Tolerance in Aquatic 
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4. Calabrese, A.J., J.R. Maclnnes, D.A. Nelson and J.E. Miller. 1977. Survival 
and Growth of Bivalve Larvae under Heavy-Metal Stress. Mar. Biol. 
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5. Clarke, G.L. 1947. Poisoning and Recovery in Barnacles and Mussels. Biol. 
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6. Crisp, D.J. and D.A. Ritz. 1973. Responses of Cirripede Larvae to Light. I. 
Experiments With White Light. Mar. Biol. 23: 327-335. 

7. Davis, J.C. 1974. Bioassay Procedures and Sublethal Effect Studies with 
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D13-D16. 

8. DeCoursey, P.J. and W.B. Vernberg. 1972. Effect of Mercury on Survival, 
Metabolism and Behaviour of Larval Uca pugilator (Brachyura). Oikos 23: 
241-247. 

9. Drummond, R.A., W.A. Spoor, and G.F. Olson. 1973. Some Short-Term 
Indicators of Sublethal Effects of Copper on Brook Trout, Salvelinus 
fontinalis. J. Fish. Res. Board Can. 30:698-701. 

10. Forward, R.B. 1977. Occurrence of Shadow Response among Brachyuran 
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287 


11. Forward, R.B. and J.D. Costlow, 1974. The Ontogeny of Phototaxis by 
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12. Forward, R.B. and J.D. Costlow. 1976. Crustacean Larval Behavior as an 
Indicator of Sublethal Effects of an Insect Juvenile Hormore Mimic. 
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13. Lang. W.H. 1976. The Larval Development and Metamorphosis of the 
Pendunculate Barnacle Octolasmis Mulleri (Coker, 1902) Reared in the 
Laboratory. Biol. Bull. 150: 255-267. 

14. Lang, W.H. 1977. The Barnacle Larvae of North Inlet, South Carolina 
(Cirripedia: Thoracica). Ph. D. Dissertation, Marine Science Program, Univ. 
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15. Mason, P.R. and P.J. Fripp. 1976. Analysis of the Movement of 
Schistosoma mansoni miracidia using Dark-Ground Photography. J. 
Parasitol. 62: 721-727. 

16. McKim, J.M. and D.A. Benoit. 1971. Effects of Long-Term Exposure to 
Copper on survival, Growth, and Reproduction of Brook Trout (Salvelinus 
frontinalis). J. Fish Res. Board Can. 28: 655-662. 

17. Olla, B.L. 1974. Behavioral Measures of Environmental Stress. In: 
Proceedings of a Workshop on Marine Bioassay, Olla, B.L. (ed.), Marine 
Tech. Soc., Washington, D.C., pp. 1-31. 

18. Percy, J.A. and T.C. Mullin. 1977. Effect of Crude Oil on the Locomotory 
Activity of Arctic Marine Invertebrates. Mar. Pollut. Bull. 8: 35-39. 

19. Pyefinch, K.A. and J.C. Mott. 1948. The Sensitivity of Barnacles and Their 
Larvae to Copper and Mercury. J. Exp. Biol. 25: 276-298. 

20. Reish, D.J., J.M. Maring, F.M. Piltz, and J.Q. Word. 1976. The Effect of 
Heavy Metals on Laboratory Populations of Two Polychaetes with 
Comparisons to the Water Quality Conditions and Standards in Southern 
California Marine Waters. Water Res. 10: 299-301. 

21. Sprague, J.B. 1970. Measurement of Pollutant Toxicity to Fish. II. 
Utilizing and Applying Bioassay Results. Water Res. 4: 3-32. 

22. Sprague, J.B. 1971. Measurement of Pollutant Toxicity to Fish. III. 
Sublethal Effects and “safe” concentrations. Water Res. 5: 245-266. 


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23. Stebbing, A.R.D. 1976. The Effects of Low Metal Levels on a Clonal 
Hydroid. J. Mar. Biol. Assoc. U.K. 56: 977-994. 

24. Vernberg, W.B., P.J. DeCoursey, and J. O’Hara. 1974. Multiple 
Environmental Factor Effects on Physiology and Behavior of the Fiddler 
Crab, Uca pugilator. In Pollution and Physiology of Marine Organisms, 
Vernberg, F.J. and Vernberg, W.B. (eds.), Academic Press, New York, pp. 
381-425. 

25. Visscher, J.P. and R.N. Luce. 1928. Reactions of Cyprid Larvae of 
Barnacles to Light with Special Reference to Spectral Colors. Biol. Bull. 
54: 336-350. 

26. Weiss, C.M. 1947. The Comparative Tolerances of some Fouling Organisms 
to Copper and Mercury. Biol. Bull. 93: 56-63. 

27. Welsh, J.H. 1932. Temperature and Light as Factors Influencing the Rate 
of Swimming of Larvae of the Mussel Crab, Pinnotheres maculatus Say. 
Biol. Bull. 63: 310-326. 

28. Young, L.G. and L. Nelson. 1974. The Effects of Heavy Metal Ions on the 
Mortility of Sea Urchin Spermatozoa. Biol. Bull. 147: 236-246. 


289 


USE OF A LABORATORY PREDATOR-PREY TEST 
AS AN INDICATOR OF SUBLETHAL 
POLLUTANT STRESS 


Christopher Deacutis 
U.S. Environmental Protection Agency 
Environmental Research Laboratory 
Narragansett, Rhode Island 02882 


ABSTRACT 

A method is presented to quantify the effects of sublethal stress on newly 
hatched and older ichthyoplankton using predation vulnerability as a 
measurable parameter. A laboratory predator-prey system was developed and 
tested using sublethal thermal shock (10°C above ambient water temperature) 
as the stressing factor. Fundulus majalis was chosen as the predator and larvae 
of Menidia menidia and Paralichthys dentatus as prey organisms. Predation 
interactions were quantified by recording all attacks, escapes, and captures, 
allowing comparison of escape probabilities (no. escapes/ attack) for control 
and shocked prey groups. 

Predator escape ability of four and six week old larvae M. menidia was 
significantly impaired following a 15 minute, +10°C thermal shock in summer 
(thermal test exposure = 30.0°C). Newly hatched and two week old shocked 
M. menidia were not significantly different from controls. Tests with P. 
dentatus showed an increase in total number of escapes following 10°C 
thermal shock in late fall tests (thermal test exposure = 25.2°C). 

The potential for laboratory predator-prey tests as behavioral bioassays to 
assess sublethal pollutant stress is evaluated, with consideration given to the 
several techniques developed to date. 

INTRODUCTION 

The present study was undertaken to develop a laboratory predator-prey 
test system to evaluate relative ecological fitness of larval fish following a 
sublethal pollutant stress. Thermal shock was employed in this case. Behavioral 
bioassays are considered to be more sensitive indicators of low-level stress in 
comparison with mortality bioassays (22). Hence, behavioral tests should serve 
to identify the less comspicuous, but nonetheless important limiting effect that 
real-world sublethal stress can have on organisms. In the case of laboratory 


290 


predator-prey tests, changes in prey escape success serve to indicate changes in 
ecological fitness, which can affect natural mortality rates in localized 
populations. 

MATERIALS AND METHODS 

For this study, larval prey species were restricted to those available from 
laboratory culture. Wild ichthyoplankton were not considered because of the 
potential for damage due to capture methods, and the difficulty in acquiring 
adequate numbers of a single species of the same age. The cultured larval prey 
species used were Menidia menidia and Paralichthys dentatus. Six hatched lots 
500/lot) of M. menidia were reared to six weeks of age and tested during 
this period. Studies with P. dentatus were limited to newly hatched larve only, 
as this species experiences high mortality at time of first feeding. All larvae 
were reared at the prevailing Narragansett Bay water temperature (M. menidia , 
summer—20.5 ±0.7°C;/ > . dentatus, late fall—15.1 ± 0.8°C). 

An attempt was made to correlate prey and predator species. Fundulus 
majalis , a carnivorous near-shore predator, was chosen as a spatially coexisting 
predator of estuarine larval fish (6). Larvae of P. dentatus are not highly 
correlated with near-shore predators since they are usually found offshore at 
hatching. However, this species was utilized to provide a larval fish with 
different swimming abilities. Paralichthys dentatus relies on high fecundity for 
successful development and eventual recruitment. Larvae of this reproductive 
strategy are usually weak-swimming relative to larvae of a species such as M. 
menidia , which has a lower fecundity, but relies on advanced morphological 
development and strong swimming capabilities at hatching. 

Biological variables controlled for this study include: reproductive condition 
of predator (a L:D 10:14 photoperiod was used to minimize reproductive 
development interference); nutritive condition (all predators were fed a mixed 
daily diet of Tetramarin flake food and adult frozen Artemia salina until 48 
hours prior to a test); predator size in relation to prey size (preliminary tests 
indicated selection of a predator size of 6-8 cm total length (TL)); feeding 
periodicity (all tests were performed at the same time of day); and hunger state 
(all predators were starved 48 hours prior to a test). 

Forty-eight hours prior to a test, each predator was placed into an 
experimental predation tank, which consisted of a polypropylene tube 30 cm 
diameter x 12.5 cm deep, with a clear Plexiglas bottom (Figure 19-1). Test 
tanks received a continuous flow of filtered seawater (y 400 ml/min.) pumped 
directly from Narragansett Bay. All predation tests occurred between 1300 and 
1500 hours at ambient bay water temperatures. 


291 


I 



B 



Figure 19-1. Predation tank and inflow funnel (A) and (B) 
Observation Bench, front and side view (normally covered 

with black plastic). 

Thermal Shock Procedures 

Larval Menidia menidia were transferred in 100 ml polypropylene beakers 
with a horizontal slit 1 cm x 2 cm wide cut 1 cm above the bottom, and 
covered with 240 u nylon screening. Larvae were placed into the beakers in 
groups of 10, four hours prior to a test and maintained at ambient water 
temperature (20.5 ± 0.7°C). For the shock tests, a seawater bath was preheated 


292 





















































to 30.0 ± 0.6°C ( A T = 9.7 ± 0.7°C) and beakers were placed into the heated 
water at six minute intervals. Containers were aerated throughout the shock 
procedure. As a 15 minute exposure period was completed, a beaker was 
immersed in seawater 1°C above ambient for a five minute cooling period. The 
larvae were introduced into predation tanks via a funnel filled with incoming 
seawater (Figure 19-1). Larvae were added at six minute intervals, and most 
larvae were eaten by the predator within the first three minutes following 
introduction. All control larvae were treated in the same manner as shocked 
larvae, but with transfer containers held at ambient water temperature rather 
than a higher temperature. 

Larvae of Paralichthys dentatus are prone to damage in screened beakers 
because of weak swimming ability and great sensitivity to handling (Grace 
MacPhee, personal communication). Therefore, intact 100 ml polypropylene 
beakers were used as transfer vessels. Ambient bay water temperature during 
these tests was 15.1 ± 0.8°C. Groups of 10 larvae were shocked by gently 
pouring the contents of each 100 ml beaker into a glass culture bowl (12.5 cm 
dia.) containing 100 ml of seawater preheated to 25.2 ± 0.8°C ( A T = 10.1 ± 
0.6°C). After the 15 minute exposure period, larvae were siphoned into the 
predation tank using silicon tubing (9.5 mm dia.). Introductions of larvae to 
the thermal treatment were again staggered at six minute intervals, as with M. 
menidia. Control larvae were treated in the same manner as shocked larvae, but 
with transfer to 100 ml of seawater at ambient water temperature. 

Quantifying Predator-Prey Interactions 

During the predation tests, all attacks, captures, and escapes were observed 
from below and recorded using an Esterline-Angus event recorder. The best 
visual field for recording observations was achieved by placing two opposing 
light sources (two fluorescent bulbs) above, yet just outside of the visual range 
of an observer directly below the tanks, and placing a flat black background 
over the tanks (Figure 19-1). This system permitted accurate recording of 
predator-prey interactions involving organisms as small as four mm. 
Significance of changes in escape probabilities, expressed as no. escapes/attack, 
were tested using the Wilcoxon distribution-free rank sum test (13). 

RESULTS 

Results of predation tests for Menidia menidia are grouped by prey age 
categories (Table 19-1). The two oldest larval groups of M. menidia (four week 
and six week old) experienced a significant decrease (P < .01) in the number of 
attacks, escapes and escapes/attack for shock tests relative to control groups. 
The two youngest age groups of this species (newly hatched and two week old) 
did not show significant treatment differences in any of the parameters 

measured. 


293 


Larval Paralichthys dentatus of only one size class (4 mm TL) were tested. 
The results stand in contrast to the findings with older Menidia larvae. A 
significant increase in escape ability (P < .01) occurred in P. dentatus larvae 
following thermal shock (Table 19-1). 

DISCUSSION 

Relationship of Results to Upper Thermal Limits 

Results of tests with four and six week old M. menidia indicate a possible 
adverse effect following an acute thermal increase to 30.0°C from an 
acclimation temperature of 20.5°C. The magnitude of this thermal elevation is 
close to the one hour TLM value of 31.4°C given by Hoff and Westman (14) 
for juvenile M. menidia acclimated to 20°C. The present study points to the 
increased sensitivity of behavioral stress indices to monitor effects of 
short-term or low level pollutant stress. Indeed, these findings strongly contrast 
the view of Austin et al ( 1). Based on mortality studies of a 13 minute shock of 
14°C above a 20°C acclimation temperature, he concluded that this treatment 
would not have any important effects on survival of larvae of this species. 

The absence of significant differences in escapes in newly hatched and two 
week old stressed M. menidia vs. the controls may be real, or could be due to 
the low number of tests run and the high variability observed within the shock 
groups. More data are necessary before final conclusions can be made on the 
sensitivity of these younger larvae to thermal shock. 

The increase in escape probability following thermal shock with larvae of P. 
dentatus may be due to an increase in altertness or in frequency of locomotory 
movements. Because the ambient water temperature was lower in tests with P. 
dentatus (15.1°C), the thermal shock did not approach reported lethal levels 
(32.0°C CTM at 15°C acclimation, Hoss et al (25)). Increased escape ability 
has been reported by Coutant (8) in juvenile Salmo gairdneri when thermal 
shock temperatures are well below lethal levels. 

Potential Mechanisms of Thermal Shock Effects on Predator 
Avoidance 

Although the phenomenon of differential predation in thermally shocked 
fish is now well documented (8, 25, 27), causal mechanisms for changes in 
vulnerability following thermal shock are not known. It has been demonstrated 
that the central nervous system is highly sensitive to temperature fluctuations 
(4, 23). The thermal sensory receptors are believed to consist of cutaneous free 
nerve endings (3), yet behavioral response to thermal shock is not necessarily 
limited to free nerve endings. Blood chemistry, membrane permeability, and 


294 


Table 19-1. Results of Predation Tests: Pooled Data for 
Each Age Category (Mean and 1 S.D.) 




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other physiological effects have all been demonstrated to occur following 
thermal shock, and may cause behavioral changes (23). Laudien (18) and 
Murray (21) both note that the lateral line system in fish is highly sensitive to 
rapid temperature changes. It is possible that disruption of normal lateral line 
function may potentially decrease response to a predator’s attack. Blaxter (7) 
considers the free neuromast system in larvae to play an important role in 
avoiding predation. 

There is evidence that the lateral line may indeed be disrupted by a thermal 
shock. Dijgraaf (10) demonstrated that the spontaneous discharge frequency 
varies with temperature in the lateral line of Xenopus (Amphibia). Murray 
(20), also working with Xenopus , noted that a sudden temperature increase of 
10°C would decrease or even completely inhibit the spontaneous discharge 
frequency, followed by compensation back to normal levels. Sudden cooling 
would cause a sudden increase in frequency. If free neuromast and developed 
lateral line receptors of fish larvae react similarly to those of Xenopus 
following thermal shock, there are two periods when the normal receptor 
frequency would be altered and signal information from the system possibly 
masked or inhibited. The first would occur upon contact with water of 
increased temperature. Thus, upon interception with a thermal discharge, and 
if the temperature differential is high enough, complete inhibition may occur, 
cutting off all signals from the lateral line momentarily. Inhibition of 
spontaneous discharge probably does not pertain to the present study, since 
predators are absent during the initial 15-minute thermal shock period. 
However, this initial neural inhibition could render larvae which pass through a 
thermal discharge plume more vulnerable to predation. Next, following such a 
thermal shock, the larvae experience rapid cooling, which could result in a 
sudden increase in lateral line discharge frequency and possible distortion or 
masking of near-field environmental stimuli. This latter effect may be involved 
in the present study since cooling of larvae occurs just prior to the predation 
interaction. 

Evaluation of Laboratory Predator-Prey Tests as Sub-lethal 
Indicators 

Laboratory predator-prey tests, such as the one described here, can be 
valuable as a means of observing subtle, but ecologically significant effects of 
low pollutant levels. In developing such tests, it is important to evaluate the 
strengths and limitations inherent in laboratory techniques utilized by other 
investigators (2, 8, 11, 12, 16, 25, 27). One must consider which primary 
predation factors are being measured by each method. Bams (2) states that a 
differential predation situation is determined by three primary factors: 
discovery rate of the prey by the predator; attack rate on the prey; and capture 
rate of the prey. 


296 


Discovery rate is assumed to be approximately equal in all methods cited 
here since exposure of all prey groups to the predators is complete and equal. 
This parameter is best measured by recording the reactive distance to the prey 
item (Beukema, 5), a difficult task which is not addressed in any of the above 
techniques. 

Attack rate can influence differential predation rate in that certain 
characteristics of the prey may be perceived by the predator and produce 
active selection. This behavior could occur in predation tests where 
simultaneous introduction of treated and control prey groups occur, as in the 
methods employed by Bams (2), Coutant (8), and Kania and O’Hara (16). 
However, as Bams noted, this parameter cannot be quantified by these 
methods because group identity of individual prey is not discernible during 
attacks. 

Differences in capture rate between prey groups are a result of differences in 
prey ability to evade an attacking predator. The techniques used by Bams (2), 
Coutant (8), and Kania and O’Hara (16) cannot discern between differences in 
capture rate and differences in attack rate since the overall result of predation 
is measured, and not individual attacks and captures. It is in this regard that the 
method devised by Yocum and Edsall (27) is superior, because it specifically 
measures the actual instantaneous predation rate as affected by changes in the 
rate of capture. Although Sylvester (25) also measured rate of capture (in 
terms of mean survival time), Yocum and Edsall found excessive variance 
between predator groups in time-to-capture when using his method. Of the 
various techniques, those of Yocum and Edsall (27), Bams (2) and Coutant (8) 
are considered best suited for laboratory predator-prey studies. The technique 
used by Kania and O’Hara (16), is similar to that of Coutant and Bams, but 
with the addition of an escape area. This modification limits its application to 
those prey species with a specialized behavioral characteristic of shallow water 
refuge. Also, there is a probable complication of learning, as prey become 
familiar with the predator’s area, and the “safe”, shallow, screened area used in 
the tests. Finally, the 60 hour test duration is fairly lengthy. 

In this particular study with larval fish prey, Bams’ (and Coutant’s) method 
of simultaneous presentation of prey from different treatment groups could 
not be utilized, as a tag to distinguish prey treatment groups is necessary. 
Common methods for identification, such as fin clipping and cauterization 
branding, were not feasible with larval prey. A visual dye is unacceptable 
because of potential alteration to predator-prey relationships due to 
conspicuousness of prey and color preferences in the predator. A number of 
fluorescent dyes were tested, but successful dyes were found to alter normal 
behavior in fish larvae (Pseudopleuronectes americanus). Efforts to label fish 
larvae with a radioisotope were also unsuccessful. Due to these difficulties 


297 


Yocum and Edsall’s technique of recording individual attacks, captures, and 
escapes was adopted. 

Both methods have intrinsic advantages and disadvantages. Barns’ method 
allows groups of predators to select between treated and untreated prey 
simultaneously. However, the test statistic used by Bams is a biased estimate of 
instantaneous mortality rate (2, 8). The method used by Yocum and Edsall 
records the actual instantaneous mortality rate in terms of attacks, escapes, and 
captures. These parameters allow a more accurate representation of changes in 
escape capabilities. In this latter method, prey treatment groups are separated, 
and the predator does not simultaneously compare prey groups. In a thermal 
plume area, where predators have been observed to attack thermally-shocked 
prey (8), it is not likely that shocked and unshocked prey would be in close 
proximity to one another. However, in studying the effects of other pollutants, 
simultaneous comparison of prey behavior may be an important factor in 
differential predation, and should be considered. 

A number of biological variables which should be considered in designing a 
laboratory predator-prey test system, including coexistence of predator and 
prey in nature (spatial and seasonal); plausible prey-predator relationship; size 
relation prey-predator; reproductive condition of predator; nutritive condition 
of predator and prey; feeding periodicity of predator; and hunger state of 
predator. Control of many of these variables has already been described in the 
Methods section. Perhaps one of the most difficult to control is satiation 
(hunger state) of the predator. Satiation state may affect prey risk (5). If a 
predator is less motivated to eat, attack efficiency may not be as high, thus 
artificially increasing escape rate of prey as measured by Yocum and Edsall’s 
method. Prey size will affect time to satiation in a predator, and must, 
therefore, also be controlled. Optimal prey size can be estimated by calculating 
a prey thickness to predator mouth size ratio with 0.5 as optimal (17, 26). 
However, even with an optimal prey size and a set deprivation schedule, 
individual variability is often substantial. Procedures to categorize motivation 
state of the predator are recommended for laboratory predator-prey tests in 
order to eliminate this variability. In the present study, the total mean number 
of larvae captured per test was calculated for all tests within each prey age 
group. A minimum percentage of this mean was chosen as an indicator of 
adequate feeding motivation. A 75 percent limit described a minimum level of 
22 larvae captured in tests with four week old M. menidia. Because six week 
old larvae were larger, the capture minimum was narrowed to 80% of the mean 
total larvae captured, giving a lower limit of eight larvae. All tests in which this 
minimum capture level was not reached were excluded from statistical 
treatment of data. In tests with larvae younger than four weeks, predators did 
not reach satiation before completion of the test, and establishment of a 
minimum capture level was unnecessary. 


298 


Laboratory predator-prey testing techniques should prove to be a useful 
tool in future pollution research. As noted, the various techniques available 
offer different approaches to the question of changes in prey vulnerability. The 
relative merits of each must be weighed with due consideration to the normal 
ecology of the predator and prey utilized, and the biological variables which 
must be controlled. 

ACKNOWLEDGEMENTS 

I would like to thank both Dr. Don C. Miller who suggested this project, and 
the staff of the EPA, Environmental Research Laboratory for their cooperation 
and helpful suggestions. I give special thanks to Miss Elaina Kenyon for her 
invaluable assistance. This work was completed in partial fulfillment of the 
requirements for Master of Science, Univ. Rhode Island. 


REFERENCES 

1. Austin, H.M., A.D. Sosnow, and C.R. Hickey, Jr., 1975. The Effects of 
Temperature on the Development and Survival of the Eggs and Larvae of 
the Atlantic Silverside, Menidia menidia. Trans. Am. Fish. Soc. 
104:762-765. 

2. Bams, R.A., 1967. Differences in Performances of Naturally and 
Artificially Propogated Sockeye Salmon Migrant Fry, as Measured with 
Swimming and Predation Tests. J. Fish Res. Board Can. 24:1117-1153. 

3. Bardach, J.E., 1956. The Sensitivity of the Goldfish (C. auratusL.) to Point 
Heat Stimulation. Am. Nat. 90:309-317. 

4. Battle, H.I., 1926. Effects of Extreme Temperature on Muscle and Nerve 
Tissue in Marine Fishes. Trans. R. Soc. Can. 20:127-143. 

5. Beukema, J.J., 1968. Predation by the Three-Spined Stickleback 

(i Gasterosteous aculeatus ): the Influence of Hunger and Experience. 
Behavior 31:1-126. 

6. Bigelow, H.G. and W.C. Shroeder. 1953. Fishes of the Gulf of Maine. U.S. 
Fish. Wildlife Serv. Fish. Bull. Vol. 53 #74, 577 pp. 

7. Blaxter, J.H.S., 1969. Development: Eggs and Larvae. In: W.S. Hoar and 
D.J. Randall (eds), Fish Physiology, Vol. III. Academic Press, New York, 

pp. 177-241. 


299 


8. Coutant, C.C., 1973. Effect of Thermal Shock on Vulnerability of Juvenile 
Salmonids to Predation. J. Fish. Res. Board Can. 30:965-973. 

9. Deacutis, C., 1977. Changes in Predation Vulnerability Following a 
Sublethal Thermal Shock on Two Species of Larval Fish. M.S. Thesis, Univ. 
of Rhode Island, Kingston, R.I. 90 p. 

10. Dijgraaf, S., 1962. The Functioning and Significance of the Lateral-line 
Organs. Biol. Rev. 38:51-105. 

11. Farr, J.A., 1977. Impairment of Antipredator Behavior in Palaemonetes 
pugio by exposure to Sublethal Doses of Parathion. Trans. Am. Fish. Soc. 
106(3):287-290. 

12. Goodyear, C.P., 1972. A Simple Technique for Detecting Effects of 
Toxicants or Other Stresses on a Predator-Prey Interaction. Trans. Am. 
Fish. Soc. 101:367-370. 

13. Hollander, M. and D.A. Wolfe, 1973. Nonparametric Statistical Methods. 
John Wiley & Sons, New York, 503 p. 

14. Hoff, J.G. and J.R. Westman, 1966. The Temperature Tolerance of Three 
Species of Marine Fishes. J. Mar. Res. 24:131-140. 

15. Hoss, D.E., W.F. Hettler, Jr., and L. Coston, 1974. Effects of Thermal 
Shock on Larval Estuarine Fish-Ecological Implications with Respect to 
Entrainment in Power Plant Cooling Systems. In: J.H.S. Blaxter (ed.), The 
Early Life History of Fish, Springer-Verlag, Berlin, pp. 357-371. 

16. Kania, H.J. and J. O’Hara, 1974. Behavioral Alterations in a Simple 
Predator-Prey System due to Sublethal Exposure to Mercury. Trans. Am. 
Fish. Soc. 103:134-136. 

17. Kislalioglu, M. and R.N. Gibson, 1976. Prey Handling Time and its 
Importance in Food Selection by the 15-Spined Stickleback, S. spinachia. 
J. Exp. Mar. Biol. Ecol. 25:115-158. 

18. Laudien, H., 1973. Activity, Behavior, etc. Ch. IV. In: H. Precht (ed.), 
Temperature and Life, Springer-Verlag, Berlin, pp. 441-469. 

19. Marine Research, Inc. 1971-1976. Brayton Point Investigations, Quarterly 
Progress Reports for the New England Power Co., Marine Research Inc., 
Falmouth, MA. 


300 


20. Murray, R.W., 1956. The Thermal Sensitivity of Lateralis Organs of 
Xenopus. J. Exp. Biol. 33:798-805. 

21. Murray, R.W., 1971. Temperature Receptors, Ch. 5. In: W. Hoar and D. 
Randall (eds.), Fish Physiology, Vol. V, Academic Press, New York pp. 
121-133. 

22.011a, B.L., 1974. Behavioral Measures of Environmental Stress. In: Olla, 
B.L. (ed.), Proceedings of a Workshop on Marine Bioassays, Marine Tech. 
Soc., Washington, D.C. pp. 1-31. 

23. Prosser, C.L., 1973. Temperature. Ch. 9. In: C.L. Prosser (ed.), 
Comparative Animal Physiology, 3rd ed., W.B. Saunders, Co., Philadelphia, 
pp. 362-428. 

24. Schubel, J.R., 1974. Effects of Exposure to Time-Excess Temperature 
Histories Typically Experienced at Power Plants on the Hatching Success of 
Fish Eggs. Estuarine Coastal Mar. Sci. 2:105-116. 

25. Sylvester, J.R., 1972. Effect of Thermal Stress on Predator Avoidance in 
Sockeye Salmon. J. Fish. Res. Board Can. 29:601-603. 

26. Werner, E.E., 1974. The Fish Size, Prey Size, Handling Time Relation in 
Several Sunfishes and Some Implications. J. Fish. Res. Board Can. 
31:1531-1536. 

27. Yocum, T.G. and T.A. Edsall, 1974. Effect of Acclimation Temperature 
and Heat Shock on Vulnerability of Fry of Lake Whitefish (Coregonus 
clupeaformis) to Predation. J. Fish. Res. Board Can. 31:1503-1506. 


301 


BURROWING ACTIVITIES AND 
SEDIMENT IMPACT OF NEPHTYS INCISA 


Wayne R. Davis 
Don C. Miller 

Environmental Research Laboratory 
U.S. Environmental Protection Agency 
Narragansett, R.l. 02882 


ABSTRACT 

It is suggested here that benthic deposit feeders are an important faunal 
group contributing to the flux of materials, including pollutants, between the 
benthos and overlying water. The present study has documented the burrowing 
and feeding activities of one dominant deposit feeder, the polychaete worm, 
Nephtys incisa, at a series of test temperatures spanning the annual thermal 
range (0-24°C) of Narragansett Bay, R.L New burrow development and feeding 
are coupled events as the worm penetrates and ingests sediment. Each new 
burrow is usually continuous with recently abandoned burrows, which results 
in extensive perforation of the benthic sediment. Then as Nephtys ventilates its 
burrow for respiratory purposes, sediment oxygenation along the entire 
subsurface burrow network also occurs. Rate of new burrow building ranges 
from one/20 days at 0°C to one/day at 24°C. 

It is hypothesized that Nephtys burrowing, feeding and irrigation activity 
contributes significantly to substrate conditioning for development of the 
aerobic benthic compartment. Doubtless, pollutant diagenesis is also directly 
influenced by this creation of an oxidative environment, resulting in significant 
pollutant fluxes to and from the benthos. 

INTRODUCTION 

The polychaete worm, Nephtys incisa , is common in silty-clay sediments of 
the northern Atlantic estuarine and coastal waters. Its dominance in fine 
sediment is a unique departure from other Nephtys species, all reported to be 
active carnivores inhabiting poor to well-sorted sands (Clark, 1962; Clark et al, 
1962). To better understand the anomalous, silty-clay habitat preference of TV. 
incisa, information regarding its in-sediment activities was pursued, primarily 
through the use of laboratory microcosms. 


302 


Soft-bodied organisms that burrow into the sediment generally do so for 
predator avoidance, at the minimum. Those which burrow continuously 
through the sediment, such as the Nephteidae, Nuclionid bivalves and 
Haustorid amphipods, do so- to obtain food either as predators or 
deposit-feeders. This vagile or wandering mode of life requires adaptations, not 
only for burrowing, but also for obtaining sufficient food and dissolved oxygen 
in this environment. The specific questions that have been investigated concern 
adaptations for burrowing, feeding and irrigation in N. incisa. The present 
paper will address certain questions of burrowing activities, while the two 
subsequent papers (Davis, 1979 b,c) will deal with feeding and irrigation 
activities in N. incisa. These activities are interrelated in that continuous 
sediment burrowing is generally a feeding adaptation and necessitates further 
adaptation to obtain well-aerated seawater while moving through the sediment. 


N. incisa occurs in estuarine, shallow coastal waters and across the Atlantic 
continental shelf from Chesapeake Bay northward to Nova Scotia, Greenland 
and Iceland, and along the European coast from the North Sea, the Baltic Sea, 
and south into the Mediterranean (Pettibone, 1963; Thorson, 1946; Bellan, 
1969). Reported population densities include 600/m 2 in Long Island Sound 
(Sanders, 1956), 300-600/m 2 in Narragansett Bay (Davis, Phelps and Morrison, 
unpublished), and up to 1500/m 2 in Buzzards Bay (Sanders, 1960). Population 
age structure has been examined temporally by Sanders (personal 
communication) who has observed three and sometimes four year classes, with 
each new year class appearing as 2 mm worms during early spring. Density of 
N. incisa can be correlated with sediment clay-silt content (Sanders, 1956), 
pollution gradients (Farrington, Quinn and Davis, 1973), and possibly 
meiofauna density (Tenore et al , 1977). 


The types of burrows found among infaunal polychaetes range from totally 
permanent to highly temporary. In the case of completely vagile worms such as 
the Nephteidae, Nereidae and Glyceridae, this in-sediment wandering may lead 
to burrow galleries and multiple openings to the surface. This mode of 
burrowing is generally adapted to exploit debris or prey on the sediment 
surface while minimizing exposure to predators. Two or more openings to the 
water also permit efficient one-way irrigation to obtain dissolved oxygen. 
Glycera alba produces such a gallery, using various burrow openings as prey 
vibration conduits and will even intercept moving prey at other gallery 
openings (Ockelman and Vahl, 1970). Nereis diversicolor has a similar gallery 
to better exploit debris on the sediment surface. An interesting adaptation for 
secondary filter feeding in this species was described by Harley (1950). Under 
certain conditions the respiratory irrigation stream is directed into a mucous 
funnel which is then eaten. Other vagile polychaetes burrow to exploit 
subsurface organic-containing sediments, for example the Capitellidae, 
Maldanidae and Paraonidae. The capitellid Heteromastus filiformis develops 


303 


semipermanent vertical burrows to reach deeper sediment in which it 
continually burrows and deposit-feeds. Oxygen exchange occurs near the 
sediment-water interface using modified posterior segments when the worm 
surfaces to defecate (Linke, 1939 ).Paraonis spp. continually burrow to form a 
deposit-feeding ring within a single stratum of high organic content (Gripp, 
1927, cited in Schafer, 1962). When the concentric burrowing reaches 8-10 cm 
in diameter, the worm burrows to a new stratum to begin another feeding ring. 

Partially or non-vagile families such as the Arenicolidae and Chaetopteridae 
possess U-shaped burrows and irrigate for the dual purposes of feeding and 
respiratory exchange. Families with the least sediment contact, termed 
tubicolous polychaetes, include the Sabellidae, Onuphidae and Serpulidae. 
These worms develop permanent tubes lined with mucopolysaccharides, shell 
debris, sand grains or calcite. They generally feed and ventilate above the 
sediment-water interface. There are many exceptions; for instance, the 
tubicolous Pectinariidae drag their sand grain tube horizontally through the 
sediment using it as an irrigation tube to the surface as they deposit-feed 
below. 

The present investigation provides information on the microhabitat of N. 
incisa by describing the nature of its burrow and examining some of the 
variables influencing burrowing. Employing laboratory in situ microcosms, 
coupled with direct observation in the field for verification, this investigation 
has addressed such topics as the form and make-up of the N. incisa burrow, 
how it is constructed, what is its horizontal and vertical extent, and how the 
rate of burrowing is influenced seasonally as the worm is exposed to 
temperatures which can range from 0-24°C. 

MATERIALS AND METHODS 

Nephtys incisa used in this study were collected from a station north of 
Conanicut Island, Narragansett Bay, Rhode Island (Figure 20-1). This benthic 
station is characteristic of a large portion of the Narragansett Bay, where a 
clayey-silt sediment covers 60-75 percent of the Bay bottom (McMasters, 
1960). Previous studies (Davis, et al ., unpublished) found N. incisa density to 
drop off rapidly in the sandy sediment toward the Bay mouth and decrease 
gradually toward the northern head of the estuary, the latter possibly due to a 
pollution gradient (Farrington, et al., 1973). 

N. incisa were collected by gently sieving Smith-Mclntire grab samples and 
by SCUBA diver-collected box cores, which were then transported intact to the 
laboratory and held in flowing seawater. When temperature change was 
required, it was shifted at a rate of 2°C per week, which is comparable to the 
rate of temperature change in the field (Figure 20-2). 


304 


MASSACHUSETTS 



Figure 20-1. 


Narragansett Bay, Rhode Island, Collection Site. 


305 






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32 

30 

28 




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1970 1971 1972 1973 


Figure 20-2. Narrangansett Bay bottom water salinity, dissolved 
oxygen and temperature (monthly measurements at sampling 
site, V 2 mile north and southwest of site). 


NOTE: Each circle represents a single measurement made on a single day (3 
replicate measurements). 


Burrowing activities were described and quantified by observing single 
worms in sediment-filled thin aquaria (2 cm thick, 14 cm wide and 15 cm 
deep). These aquaria were maintained in flow-through systems at appropriate 
temperatures. The thin aquaria allowed free three-dimensional movement 
(worms can easily turn in 1 cm thick aquaria), yet also permitted direct 
observation of some activities with a stereomicroscope when the worms 
burrowed along the glass. The glass walls are considered to represent an 
obstacle to the worm not unlike buried rocks or bivalve shells, which are 


306 






commonly observed in the field. Experimental worm density was one per 
experimental aquarium or one worm/28 cm^. The aquaria were kept in the 
dark except for a two-minute interval once weekly when both sides of the 
squaria were photographed. 

Patterns and rates of burrowing and sediment aeration were described from 
sequential photographs of the worm burrows constructed against the glass wall 
of the sediment-filled thin aquarium. Sediment aeration by local burrowing and 
irrigation was indicated by a change in sediment color from dark to light and 
provided a record of the worm’s present or past location. This method relies on 
oxygen-sediment-color relationships proposed by Fenchel and Riedl (1970), 
Hayes (1964), Teal and Kanwisher (1961), Rhoads, Aller and Goldhaber 
(1977), and Aller and Yingst (1978). The absence of oxygen generally leads to 
a dominance of reduction reactions (eH<0) including formation of iron 
sulfides which blacken the sediment. At the point of change from dark to light 
color in the sediment, values for both eH (volts) and dissolved oxygen (mg/1) 
begin to increase from 0.0. Presence of oxygen is key to substrate oxidation 
reactions (eH<0). By quantifying the development of this color discontinuity 
against the thin aquarium glass wall where worms are burrowing, it is possible 
to document three parameters of burrowing activity: 1) the spatial-temporal 
extent of burrowing, 2) the effective new surface area of the sediment-water 
interface, and 3) the extent of sediment aeration. This record, visible against 
the aquarium wall, can be photographed at appropriate time intervals and 
activity quantified by counting burrows, measuring the surface area of burrow 
linings and by planimetry, measuring the volume of aerated sediment. 
Horizontal burrowing patterns were also described by recording temporal and 
spatial appearance of new burrow openings at the sediment surface in large 
sediment-filled dishes (single .2-.3 g worms in 3 x 6 x 4” deep sediment trays). 

RESULTS 

Description of the Burrow 

Nephtys incisa actively penetrates fine sediments and establishes an 
open-ended burrow with no visible modification of the burrow wall except 
packing. It is not known if mucous, exuded onto parapodial setae during 
feeding (Davis, 1979b), is present in the burrow wall. The burrow is often 
W-shaped, but many variations exist. Back and forth motion of the worm with 
packing of the burrow wall by setal bundles creates a section which is closely 
fitted to the front and mid section of the worm. This precise fit permits 
flow-through irrigation by the parapodial cilia (Davis, 1979c). The occupied 
burrow is often continuous with a recently abandoned burrow posteriorly, 
which then continues to receive oxygenated water before it returns to the 
surface. Abandoned burrow segments gradually till with suspended particulates 


307 


and/or from the collapse of old burrow walls. There is also avalanching of 
surface floe into efferent and afferent burrow openings. The volume of 
suspended sediment transported in this manner into the deeper benthos then is 
proportional to total burrow volume. 

Method of Burrow Building 

New burrow building is accomplished as TV. incisa first penetrates the wall of 
its existing burrow with an undulating proboscis, displacing small amounts of 
sediment. Worm position for this initial step is maintained through hydrostatic 
enlargement of the posterior segments. The worm next penetrates the sediment 
by lengthening the anterior segments and finally, as the head penetration 
reaches its forward limit, the pharynx everts, creating a bulbous cavity in the 
sediment. This type of sediment penetration has been described as “bolting” 
by Schafer (1962). He states that this is a common form of sediment 
penetration and is accomplished by contracting all body musculature. The 
resulting pressure forces coelomic fluid into the anterior region, forcing those 
segments without longitudinal muscles to expand and finally everting the 
pharynx at peak pressure. When the pharynx is re-inverted, TV. incisa swallows a 
slurry of sediment which was created as the compacted sediment was 
penetrated. The whole sequence is repeated until the worm occupies the new 
burrow fully and has established a new opening to the surface. The entire 
process can usually be accomplished in less than an hour. 

Spatial Extent of Burrowing 

A series of observations were made to determine if burrowing followed 
some functional pattern vs random burrowing and also to determine the 
horizontal and vertical scope of burrowing. A typical sequence of new burrow 
formation is illustrated in Figure 20-3. This figure is a two-dimensional 
reconstruction of a three-dimensional activity which is typically only partially 
visible against the glass wall. It represents an example of burrowing but does 
not indicate a predictable pattern of burrowing. Figure 20-3 shows actual 
tracings from weekly photographs of a different burrow building sequence (a 
single worm over a six-week period at 18°C). Observations were also made with 
worms in large dishes of sediment so that horizontal movement could also be 
assessed without wall interference. Each new afferent burrow opening was 
mapped, with new openings connected as a series of vectors (Figure 20-4). At 
the time of the last recording, the worm is probably lying in a burrow between 
opening 10 and 11, with the head located toward opening 11. The magnitude 
of ea<Ji horizontal vector was found often correlating with the size of the 
worm (Figure 20-5), with burrow length approximately 2-3 times the length of 
TV. incisa. Yet exceptions do occur, as shown in Figure 20-4 by the short 
distance to afferent openings 2 and 4. By observing 30 six-week sequences of 


308 



NOTE: A. Diagram of the reconstructed sequence of new burrow building 
(burrows nos. 2 and 3) by a single worm, with collapse of original burrow (No. 
1). "A", anterior, "P", posterior; direction of movement. B. Diagram of the 
vertical burrow network developed by a single worm and its persistence over 
time (1 8°C). 


309 
































Figure 20-4. Horizontal burrowing pattern of a single Nephtys 
incisa as indicated by the sequence of new afferent burrow 
openings appearing on surface of large sediment-filled box 
over a period of 2 weeks (18°C). 


NOTE: Afferent opening no. 11 indicates most recent burrow opening. 



Figure 20-5. Relationships of size of N. incisa to burrow length 

using a flexible cm rule. 


NOTE: Each vertical bar represents the range of 4-7 observations of a single 
worm of the indicated weight. N = 7 worms. 


310 



















vertical and horizontal burrowing, it was concluded that no pattern of burrow 
gallery formation existed but that burrowing was a meandering extension of 
past burrows. 

Depth of sediment penetration likewise correlates with worm size (Figure 
20-6). The lower vertical limit of burrows for first-year worms (up to 0.1 g wet 
weight) is 2-3 cm beneath the sediment-water interface. Second-year worms 
(up to 0.4 g) limit burrowing to 7-8 cm. Three-year old worms (up to 1 g) may 
burrow as deep as 14-15 cm. N. incisa of all sizes may be found near the 


E 

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WORM WEIGHT (g WET WT) 



Figure 20-6. Nephtys incisa Mean Depth of Burrowing in 

Relation to Worm Size. 


311 










































sediment-water interface, indicating that N. incisa may perforate sediment at 
all levels down to its size-limited depth. 

Temporal Aspects of Burrowing 

Rate of new burrow formation is temperature-dependent. Figure 20-7 
illustrates both the numbers of new burrows formed per week and its 
reciprocal, duration of burrow occupancy, at Five temperatures spanning the 
annual thermal range. At 0°C, burrow turnover averages one new burrow built 
every two weeks. At 6°, an average of 1.5 burrows are built per week; 3-3.5 at 
12°; 3.5-4 at 18° and 6.5-7 burrows were observed per week at 24°C. 

BURROW FORMATION RATE (PER WEEK) 


K)OJ (Ji CT) -nI 00 CO O 



DURATION OF BURROW OCCUPANCY (DAYS) 

Figure 20-7. Relationship between Nephtys incisa burrow 
formation rate and burrow occupancy to temperature. 

NOTE: Mean and range observed indicated by -0- (range limits refer only to 
burrowing rate axis). N = 6 for each temperature level. 


312 
















Burrow-Sediment Relations 


The sediment color along present and past burrows was also useful in 
semi-quantifying the role of N. incisa in 1) aeration of sediment and 2) in 
increasing the effective surface area of the sediment-water interface into the 
benthos. There is always some oxygen diffusion across any sediment-water 
interface, assuming the overlying water is oxygenated. Wherever burrows 
penetrate the sediment and are irrigated with oxygenated water, a halo of light 
brown or yellow oxygenated sediment soon develops around the burrow. The 
transition of yellow-brown to black, 2-5 cm deep, approximates the limit of 
oxygen penetration. Thus an increase in “aerobic” sediment is described by the 
expression: 


^halo ^burrow-halo system ' ^burrow 

This volume was estimated here by measuring the light brown oxidized zone 
visible against the thin, sediment-filled glass aquaria using planimetry. This 
subsurface oxygenation persists for some time after a worm abandons the 
burrow, since efferent oxygenated irrigation water typically continues to 
course through old burrows. The rate of increase in sediment aeration 
following introduction of a single worm in a thin aquarium at 18°C is 
summarized in Figure 20-8. The 2-5 mm thick “aerobic” halo is continuous 
with and as thick as the aerobic zone at the sediment-water interface (see 
Figure 20-3). The dotted line in Figure 20-8 represents the depth of aerobic 
sediment in an aquarium without any worm present, that is, the aerobic zone 
at the sediment surface. Any increase above this level represents that resulting 
as a consequence of burrow irrigation activities. By comparing the aeration 
rates depicted in Figure 20-8, it is apparent that the slope of the curve increases 
with water temperature. This rise in sediment aeration eventually levels off in 
time as an equilibrium develops between oxygenation of new burrow sediment 
and chemical reduction of oxygenated sediment along old abandoned burrows. 
Figure 20-8 summarizes the relationship between temperature and rate of 
Nephtys sediment aeration observed in laboratory in situ thin aquaria. It is 
apparent that the extent of sediment oxygenation is positively correlated with 
temperature, i.e. more oxygen is delivered to deeper layers during warmer 
seasons. Hence, even though oxygen demand by the benthos is at its maximum 
during warm periods, the actual sediment aeration through Nephtys burrowing 
and irrigation can be even greater. 

DISCUSSION 

N. incisa burrows through sediment using adaptations previously described 
for other Nephtys and Nereis species (Schafer, 1962) and for Arenicola marina 
(Wells, 1952). This specialized locomotion called “bolting” refers to the head 


313 



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NOTE: The quantity of aerated sediment visible against the thin aquarium 
wall is expressed as mean depth of aerated sediment, although its distribution 
is related to burrow location as indicated in Figure 20-3. 

NOTE: Dotted line represents mean depth of aerobic sediment in sediment- 
filled, thin-walled aquaria in absence of benthic organisms. Mean aerobic sed¬ 
iment depth is measured through planimetry of vertical burrowing pattern (e.g. 
Figure 20-3). 


314 


































being forced into consolidated sediment.- This is accomplished by contracting 
all body muscles, creating coelomic fluid pressure to expand anterior segments. 
The hard, bullet-shaped muscular proboscis is forced into the sediment and the 
pharynx finally everted when coelomic pressure reaches its peak. At this point 
N - incisa inverts its pharynx, literally sweeping an emulsion of sediment into 
the midgut, carried by fleshy, finger-like papillae at the tip of the everted 
pharynx. Immediately the posterior region of the worm crawls into the new 
cavity through peristaltic contractions. The bolting action is then repeated 
until a new burrow to the surface is complete. Other means of polychaete 
locomotion are also used by N. incisa for sediment penetration. Body 
undulation common to nereids is typically used by N. incisa to enter sediment 
from the water column. This more rapid sediment penetration is used only 
when N. incisa lacks a sufficient anchor on the sediment. Peristaltic locomotion 
in the Capitellidae is limited to movement within a burrow cavity. Both 
undulation and peristalsis involve a wave of segmental muscle contraction along 
the body from head to tail if locomotion is directed forward (Schafer, 1962). 

Burrow maintenance by N. incisa appears limited to packing loose sediment 
against the burrow wall as observed in Nereis spp. and is termed “wallpapering” 
(Schafer, 1962). Burrow wall integrity may also be maintained by mucous 
since it is readily observed covering the setae. In addition, Schafer suggested 
that the iron oxides in the surrounding sediment (oxide halo) is itself a local 
“cementing” of sediment. 

N. incisa develops temporary burrows, but unlike the continuous burrowers 
(e.g. Paraonis, Heteromastis or Pectinaria), N. incisa rapidly completes a new 
open burrow and then remains in it from one day during the summer to three 
weeks in the winter. Such a burrowing sequence suggests that N. incisa is 
abandoning discreet burrows rather than continuously meandering. The period 
of temporary burrow residence, occupied with feeding and irrigation activities, 
will be addressed in the following two papers (Davis, 1979 b,c). 

The magnitude and orientation of new burrow construction may offer 
insight to in-sediment adaptations such as feeding or predator avoidance. In N. 
incisa the scalar values of burrowing depth and breadth appear to be strictly 
worm-size related, with larger worms burrowing increasingly deeper and 
covering greater horizontal distances. The vector measurement of sequential 
burrow direction appears to be random. This in contrast to other vagile 
polychaete burrowers. Glycera and Nereis , for instance, develop burrow 
galleries that maximize the worm’s ability to exploit large areas of sediment 
surface for prey and food debris respectively. The deposit-feeders Paraonis and 
Heteromastis burrow in patterns termed “guided meandering”. This systematic 
exploitation presumably minimizes repeated ingestion of sediment recently 
eaten. N. incisa shows no indication of such adaptations. 


315 


A general expression describing this depth-related burrowing intensity may 
be stated: 


i = 1 

0)i z = 2 



‘ B t y i + Y 2 



i = n 


where I z is the depth-related burrowing intensity, Dyj is the mean length of 
burrowing by the i^ 1 year class (from Figure 20-6), Bj is the 
temperature-dependent burrowing rate (Figure 20-7) and Y 2 is the size of the 
2nd year class. 

The influence of N. incisa on increasing the sediment-water interface surface 
area is best described by computing the surface area of the burrow wall since 
the burrow is continuously irrigated and may be stated: 

i = 1 

(2) S.A. l = 2 L Yi C Yj + C Y 2 '' ’ C Yn 
i = n 


where S.A.^ is the burrow lumen wall surface area of a population of N. incisa , 
Lyj is the mean burrow length of the Yi year class and Cy n is the mean 
circumference of the burrow of the nth year class. 

Burrow irrigation by N. incisa results in the oxidation of surrounding 
sediment. The degree of oxidation has only been expressed in a qualitative 
sense here. However, since the thickness of the sediment “halo” is virtually 
identical to the oxidized zone at the sediment-water interface, the influence of 
N. incisa on oxygenating subsurface sediment can be quantititively expressed 
by calculating the volume of light brown aerobic sediment surrounding the 
burrow for each year (size) class and extrapolating this figure over the density 
of that size class in a square meter of sediment: 


i = 1 

(3) ®N. incisa ~ ^ ^halo 
i = n 



where 0 is the quantity of oxygen-containing sediment. Vj ia j 0 is the volume of 
the oxygenated halo (Figure 20-8) and Y| is the density of the 1st year class in 
worms/m". 

The silt-clay habitat of N. incisa is unique within the genus Nepktys , 
virtually singular in its sand-dwelling, predaceous life mode. This departure in 


316 


sediment preference may be related to its equally unique deposit-feeding habit 
since silt-clay sediments are typically rich in organic matter. Motility of N. 
incisa in fine sediment can best be described as an extended period of open 
burrow habitation followed by its extension of the burrow developing another 
temporary burrow. This habit is different from infauna that constantly burrow 
as predators, or continual deposit-feeders, or those which reburrow only 
because of complete burrow destruction. Whatever the purpose(s) for high 
burrowing rates by N. incisa , reported densities of 600 — 1200 per rrT may 
significantly perforate the top 10 — 15 cm of sediment during warmer months 
(such as suggested by model #1 above). This perforation probably results in a 
large increase in the surface area of the sediment-water interface (model #2). 
Since both present and some past burrows are irrigated, this expansion of 
surface area may result in a general biological model for sediment-seawater 
exchange, assuming a gradient as in dissolved oxygen (model #3). 

REFERENCES 

1. Aller, Robert C. and Josephine Y. Yingst. 1978. Biogeochemistry of 
Tube-Dwellings: A study of the Sedentary Polychaete Amphitrite ornata. J. 
Mar. Res. 36(2):201-265. 

2. Bellan, Gerard. 1969. Contribution a l’Etude des Annelides Polychetes de 
la Region de Rovinj (Yougoslavia). Jugoslavenska Akademija Znanosti I 
Umjetnosti. pp. 25-55. 

3. Clark, R.B. 1962. Observations on the Food of Nephtys. Limnol. 
Oceanogr. 7(3):380-385. 

4. Clark, R.B., J.R. Alder and A.D. McIntyre. 1962. The Distribution of 
Nephtys on the Scottish Coast. J. Anim. Ecol. 31:359-372. 

5. Davis, Wayne R. 1979b. Feeding Activities in Nephtys incisa. Part II of 
Ph.D. Thesis, University of South Carolina. 

6. Davis, Wayne R. 1979c. Irrigation Activities in Nephtys incisa. Part III of 
Ph.D. Thesis, University of South Carolina. 

7. Farrington, John W., James G. Quinn and Wayne R. Davis. 1973. Fatty 
acid composition of Nephtys incisa and Yoldia Umatilla. J. Fish. Res. Bd. 
Canada. 30:181-185. 

8. Fenchel, T.M. and R.J. Riedl. 1970. The Sulfide System: A New Biotic 
Community Underneath the Oxidized Layer of Marine Sand Bottoms. 
Marine Biology. 7(3):255-268. 


317 


9. Harley, Margaret B. 1950. Occurrence of Filter-Feeding Mechanism in the 
Polychaete Nereis diversicolor. Nat. 165:734-735. 

10. Hayes, F.R. 1964. The Mud-Water Interface. Oceanogr. Mar. Biol. Ann. 
Rev. 2:121-145. 

11. Linke, 0. 1939. Die Biota des Jadebusenwattes. Helgol. wiss. Meeresunters. 
1:201-348. 

12. McMasters, Robert L. 1960. Sediments of Narragansett Bay System and 
Rhode Island. J. of Sedimentary Petrology. 30(2):249-274. 

13. Ockelman, Kurt and Ola Vahl. 1970. On the Biology of the Polychaete 
Glycera alba , Especially Its Burrowing and Feeding. Ophelia. 8:275-294. 

14. Pettibone, Marian H. 1963. Marine Polychaete Worms of the New England 
area. Part 1. Museum of Natural History, Smithsonian Institution. 

15. Rhoads, Donald C., Robert C. Aller and Martin B. Goldhaber. 1977. The 
Influence of Colonizing Benthos on Physical Properties and Chemical 
Diagenesis of the Estuarine Seafloor. In: Ecology of Marine Benthos. Ed: 
Bruce C. Coull. Univ. So. Carolina Press, Columbia, So. Carolina, pp. 
113-138. 

16. Sanders, Howard L. 1956. Oceanography of Long Island Sound, 
1952-1954: X. The Biology of Marine Bottom Communities. Bull. Bingham 
Oceanogr. Coll. 15:345-414. 

17. Sanders, Howard L. 1960. Benthic Studies in Buzzards Bay. III. The 
structure of the soft-bottom community. Limnol. and Oceanogr. 
5(2): 138-153. 

18. Schafer, Wilhelm. 1962. Ecology and Palaeoecology of Marine 
Environments. Oliver and Boyd. Edinburgh, Scotland. 568 p. 

19. Teal, John M. and John Kanwisher. 1961. Gas Exchange in a Georgia Salt 
Marsh. Limn, and Oceanogr., 6:388-399. 

20. Tenore, Kenneth R., John H. Tietjen, and John J. Lee. 1977. Effect of 
Meiofauna on Incorporation of Aged Ellgrass, Zostera marina , Detritus by 
the Polychaete Nephthys incisa. J. Fish. Res. Bd. Canada. 34:563-567. 


318 


21. Thorson, Gunnar. 1946. Reproduction and Larval development of Danish 
Marine Bottom Invertebrates, with Special Reference to the Planktonic 
Larvae of the Sound (Oresund). Meddelelser fra Kommissionen for 
Danmarks fiskeriog Havundersogelser. 4(1 ):523 p. 

22. Wells, G.P. 1952. The Proboscis Apparatus of Arenicola. J. Mar. Biol. Ass. 
U.K. 31:1-28. 


319 


SECOND GENERATION PESTICIDES 
AND CRAB DEVELOPMENT 

John D. Costlow and C. G. Bookhout 
Duke University Marine Laboratory 
Beaufort, North Carolina 28516 


ABSTRACT 

A number of compounds have been introduced recently as potential 
substitutes for the traditional “hard” pesticides in the control of insect 
populations. Some of these compounds, juvenile hormone mimics or analogs, 
are intended to simulate the activity of naturally occurring juvenile hormones 
and prevent metamorphosis or, in the case of insect growth regulators, control 
differentiation or specific physiological processes at specific stages of 
development. Because of the phylogenetic relationship between insects and 
crustaceans, one might legitimately expect that those compounds which alter 
or interfere with the developmental pattern of insects could also have similar 
effects on the developmental stages of marine crustaceans. 

In salinities of 20 and 35 ppt, 100 percent mortality of megalopa of C. 
sapidus occurred when exposed to 10 ppm MONO-585 while 1 ppm reduced 
survival from 100 percent to 40 percent. 100 percent mortality in the zoeal 
stages of R. harrisii was observed with a dilution of 1.0 ppm in reduced 
salinities but at 20 and 35 ppt, survival was unaffected. The concentration of 
10 ppm MONO-585 was lethal in all experimental salinities. Exposure of C. 
sapidus megalopa to 0.1 ppm Methoprene resulted in reduced survival only 
when lower temperatures (20-25°C) were used. Juvenile crab stages I through 
IV were unaffected by the concentrations of Methoprene used. 

The findings of these experiments and their possible significance to normal 
development of larvae of these two species within the natural environment are 
considered. 


INTRODUCTION 

A number of compounds have been introduced recently as potential 
substitutes for the traditional, “hard” pesticides (DDT, Malathion, Dieldrin, 
etc.) in the control of insect populations. Some of these compounds, juvenile 


320 


hormone mimics or analogs, are intended to simulate the activity of naturally 
occurring juvenile hormones and prevent metamorphosis or, in the case of a 
second group, insect growth regulators, to control differentiation or specific 
physiological processes during development. 

Several authors have reported the effects of juvenile hormone mimics on the 
development of insects (15, 17, 19 and others). Only a few studies, however, 
are reported on the effects of these compounds on other invertebrates (9, 10, 
13). Of these Gomez et al (9) and Ramenofsky et al (13) first described the 
effect of two juvenile hormone mimics on development and metamorphosis of 
the cirripede, Balanus galeatus, and Tighe-Ford (1977) subsequently reported 
juvenile hormone analog effects on another species of barnacle, Elminius 
modestus. Studies on representative species of other marine Crustacea are 
limited, but do include the effect of two juvenile hormone mimics on larval 
development of the mud-crab, Rhithropanopeus harrisii (1, 2, 4). A study by 
Forward and Costlow (8) describes the manner in which one of these 
compounds may affect the behavior of crab larvae. Pa yen and Costlow (11) 
studied the effects of juvenile hormone mimics on gametogenesis of adult 
Rhithropanopeus harrisii. 

Because of the phylogenetic relationships between insects and crustaceans, 
one might legitimately expect that those compounds which would alter or 
interfere with the developmental pattern of insects could also have similar 
effects during the development of marine decapods. 

The present study was undertaken to further explore the effects of two 
compounds, methoprene (Zoecon Corporation) and MONO-585 (Monsanto 
Corporation) on the development of larvae of estuarine crabs. Specifically, 
experiments were designed to determine if these compounds would affect 
survival of the larvae, alter the number of larval stages, change the time 
required for development of all stages and metamorphosis, or affect the 
frequency of molting within the early juvenile crab stages after metamorphosis. 
A second portion of the experiment was designed to determine if effects of 
these compounds would be altered by changes in such environmental factors as 
salinity and temperature. 

The two species which were selected for study were the small mud-crab, 
Rhithropanopeus harrisii (Gould), and the megalopa of the commercial blue 
crab, Callinectes sapidus Rathbun. 

MATERIALS AND METHODS 

Following the general rearing procedures described by Costlow and 
Bookhout (5) and Costlow, Bookhout and Monroe (7) ovigerous females of C 


321 


sapidus and R. harrisii were brought in from the waters of the Newport Estuary 
in the vicinity of Beaufort, North Carolina, and maintained in salinities and 
temperatures most closely approximating those of the experimental conditions 
until hatching of the larvae occurred. With the experiments on 
Rhithropanopeus harrisii, the larvae were set up in separate experimental series 
consisting of 50 larvae per species, and maintained in temperature controlled 
cabinets with a photoperiod of 12 hours light and 12 hours dark, until the 
fourth juvenile crab stage was reached. 

In experiments with Callinectes sapidus, which involved only the megalopa 
stage, larvae were maintained through the seven zoeal stages of 30 ppt, 25°C 
until the final zoeal molt. At that time, 20 megalopa were transferred to each 
of the experimental salinity and temperature conditions, and maintained in a 
photoperiod consisting of 12 hours light and 12 hours dark until the fourth 
juvenile crab stage was reached. Within each of the experimental series a 
control series was maintained, and at least one acetone-control series was 
maintained, since both methoprene and MONO-585, only slightly soluble in 
water, were prepared from the pure compound as an acetone stock solution of 
1 ppt. 

The two compounds used in this experiment were methoprene (Altosid^: 
ZR-515; isopropyl 1 l-methoxy-3, 7, 1 l-trimethyldodeca-2, 4-dienoate) 
manufactured by Zoecon Corporation, Palo Alto, California, and MONO-585 
(2, 6-di-t-butyl-4- (aadimethylbenzyl) phenol) manufactured by Monsanto 
Chemical Company, St. Louis, Missouri. 

In the experiments on Rhithropanopeus harrisii larvae involving MONO-585, 
dilutions of 10, 1 and 0.1 ppm were used in combination with 25°C, known 
from previous work to be the optimum temperature for development (7), and 
salinities of 5, 20 and 35 ppt. 

In experiments with Callinectes sapidus megalopa, a variety of salinities, 
constant temperatures, and cyclic temperatures were combined with the 
dilutions of MONO-585 (10, 1,0.1 ppm) or methoprene (0.1 and 0.01 ppm). 
These included, depending on the particular series, salinities ranging from 5 to 
35 ppt and temperatures, constant or cyclic, ranging from 20°C to 35°C. The 
specific conditions for individual experimental series will be considered in 
connection with the results. 

Larvae in all series were checked each day for survival and stage of 
development, the numbers being recorded for each experimental series. 
Individual megalopa were segregated in plastic compartmented boxes to avoid 
cannabalism, and also to facilitate recording the time of metamorphosis to the 
first and subsequent crab stage for each individual. 


322 


RESULTS 


Effect of MONO-585 on Development 

Survival of the zoea of R. harrisii was unaffected by the presence of 1.0 and 
0.1 ppm MONO-585 when the larvae were maintained in salinities of 20 and 35 
ppt (Figure 21-1). In the reduced salinity of 5 ppt, however, total mortality 
within the zoeal stages was observed with a dilution of 1.0 ppm, while survival 
at 0.1 ppm was higher than that observed for either the seawater control or the 
acetone control (Figure 21-1). A concentration of 10 ppm MONO-585 was 
lethal in all three experimental salinities and none of the zoeae developed 
beyond the first stage. 

Megalopa of R. harrisii were affected by the presence of 1.0 ppm 
MONO-585 when combined with a high salinity of 35 ppt but survival of this 
last larval stage was only slightly reduced at 20 ppt (Figure 21-1). There were 
no reductions in survival of megalopa in 0.1 ppm, regardless of the salinity. 

In those experimental salinities in which some development occurred, the 
time required for development from hatching to the megalopa, megalopa to the 
crab, and hatching to the time of Final metamorphosis to the crab, was 
unchanged by the presence of either 1.0 or 0.1 ppm MONO-585 (Figure 21-2). 
The development pattern followed the sequence of four zoeae and one 
megalopa normally observed for R. harrisii and no additional or supernumerary 
larvae were noted. 

As indicated in Figure 21-3, total mortality of megalopa of C. sapidus was 
observed in all series maintained at 5 ppt, including the control. In salinities of 
20 ppt and 35 ppt, 10 ppm MONO-585 resulted in total mortality. One ppm 
reduced survival from 100 percent observed in the controls to 40 percent, 
regardless of salinity, and 0.1 ppm reduced survival to approximately 90 
percent. Time for metamorphosis of the megalopa, from the final zoeal molt to 
the appearance of the first juvenile crab, varied from a mean of approximately 
8 days to 11 days, but the presence of MONO-585 did not appear to be related 
to this variability (Figure 21-4). 

When cultured in 5°C, 24 hour cyclic temperature (20-25°; 25-30°; and 
30-35°: Costlow and Bookhout, 1971) there was no significant change in 
survival of control series or those series of megalopa maintained at 0.1 ppm 
MONO-585 (Figure 21-5). There was, however, some reduction in survival of 
megalopa maintained in 1.0 ppm MONO-585 coupled with all three cyclic 
temperatures. The greatest reduction in survival occurred when the compound 
was combined with a salinity of 35.0 ppt, but this effect was reduced when the 
cyclic temperature was increased to the maximum level of 30-35°C. 


323 



Figure 21-1. Survival of Larvae of Rhithropanopeus Harrisii 
Maintained at 25°C f 5 ppt, 20 ppt and 35 ppt when Exposed to 

Three Dilutions of MONO-585. 

NOTE: C-control; AC-acetone control: 10, 1 , and 0.1 ppm-dilutions of 
MONO-585. 


324 






































































































































Figure 21-2. Time of Development for Zoeae and Megalopa of 
Rhithropanopeus Harrisii Maintained at 25°C, 5 ppt, 20 ppt and 
35 ppt, when Exposed to Concentrations of MONO-585. 

NOTE: C-control; AC-acetone control: 10, 1, 0.1 ppm-dilutions of 

MONO-585. The vertical column represents the range and the horizontal line 
the mean. 


325 















































































Figure 21-3. Survival of Megalopa of Callinectes Sapidus 
Maintained at 25°C, 5 ppt, 20 ppt, and 35 ppt f when Exposed to 

Dilutions of MONO-585. 

NOTE: C-control: 10, 1, 0.1 ppm-dilutions of MONO-585. The vertical column 
represents the range and the horizontal line the mean. 


CALLINECTES SAPIDUS 
M-0585 25 °C 


LU 

2 

Q. 

O 

J 

LU 

> 

LU 

Q 


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>- 

< 

Q 


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O 


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Figure 21-4. Time Required for Metamorphosis of Megalopa of 
Callinectes Sapidus Maintained at 25°C f 5 ppt r 20 ppt, and 35 ppt, 
when Exposed to Concentrations of MONO-585. 

NOTE: C-control: 10, 1, 0.1 ppm-dilutions of MONO-585. The vertical column 
represents the range and the horizontal line the mean. 


326 


































































CALLINECTES SAPIDUS 
M-0585 - CYCLIC TEMPERATURES 






ooooooo oooo 

O O 00 N *0 ^ rocsj — 


01 -W 


IVAIAUflS ±N30U3d 



327 


Figure 21-5. Survival of Megalopa of Callinectes Sapidus 
Maintained at Three Cycles of Temperature (20-25°C, 25-30°C, 
and 30-35°C), Three Salinities (15 ppt, 25 ppt # and 35 ppt) 
when Exposed to Two Dilutions of MONO-585. 









































CALLINECTES SAPIDUS 
M-0585 - CYCLIC TEMPERATURES 


DAYS OF DEVELOPMENT 

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9 

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- n* n 

u _a_ 

15%. 

25%. 

— | 

Ippm Olpprn C Ippm Olppm C Ippm 0 1 ppm 

20-25*C 25-30*C 30-35*C 


Figure 21-6. Time Required for Metamorphosis of Megalopa of 
Callinectes Sapidus (M-1C) and Subsequent Juvenile Molts 
(1C-2C, 2C-3C, 3C-4C, and 4C-5C) when Maintained at 
Three Cycles of Temperature (20-25°C, 25-30°C, 
and 30-35°C), Three Salinities (15 ppt, 25 ppt, and 35 ppt) 
when Exposed to Two Concentrations of MONO-585. 

NOTE: The vertical column represents the range and the horizontal line the 
mean. 


328 








































































Once the megalopa have metamorphosed to juvenile crabs, there is no 
significant effect on survival during the subsequent four juvenile molts due to 
either salinity or the presence of 1.0 ppm or 0.1 ppm MONO-585 (Figure 
21-6). The time required to complete the individual molts (first crab to second 
crab, second crab to third crab, third crab to fourth crab, and fourth crab to 
fifth crab) varies considerably in all experimental series (Figure 21-6). As might 
be expected, these same intervals were considerably reduced when the 
megalopa and juvenile crabs were maintained in the cyclic temperatures of 
25-30°C and 30-35°C (Figure 21-6). 

Effect of Methoprene on Development 

Neither the megalopa nor the early juvenile crabs of C. sapidus 
demonstrated significant changes in survival when exposed to 0.1 or 0.01 ppm 
dilutions of methoprene combined with salinities of 15, 25 and 35 ppt, and 
maintained in cyclic temperatures of 25-30°C or 30-35°C (Figure 21-7). In the 
reduced temperature cycle of 20-25°C, however, survival was reduced from 

20- 25 percent in the presence of 0.1 ppm methoprene, in all salinities. Juvenile 
crab stages, one, two, three, and four, however, did not show any reduction in 
survival when maintained in these same combinations. In the same 
combinations of cyclic temperature, salinity, and methoprene, there is no 
apparent change in time required for completion of the megalopa stage or in 
the interval periods observed for the first and subsequent crab stages (Figure 

21 - 8 ). 


DISCUSSION 

The relatively few studies to date on the effect of insect growth regulators 
on marine Crustacea have demonstrated that one may expect a variety of 
effects, depending upon the species and the chemical compound itself. Gomez 
et al (9) and Ramenofsky et al ( 13) found that hydroprene (Altozar R ) caused 
premature metamorphosis of larvae of the barnacle B. galeatus while a second 
mimic, methoprene (Altosid R ) had no effect on the time of metamorphosis, 
nor did it prevent settling when a proper substrate was available. 

Two other analogs, farnesyl methyl ether (FME) and ethyl, 10, 11-epoxy-3, 
7, 10, 1 l-tetramethyl-2-cis-trans-6-cis-trans-dodeca-dienoate (Ro-8-4314) were 
shown by Tighe-Ford (1977) to interfere with the development of Elminius 
modestus larvae, with the effect apparently related to the state of physiological 
development of larvae at the time of exposure. Costlow (4) described the 
effects of methoprene (Altosid R ) on larvae of the estuarine mudcrab, 
Rhi thro panop eus harrisii (Gould) and indicated that 1.0 ppm resulted in total 
mortality of the larval stages, usually within the First two days of hatching. If 
the larvae were maintained in salinities as low as 5 ppt, survival within the 


329 



or 

=> 

< 
CO cc 
3 UJ 
Q CL 

0- S 

< UJ 
CO h- 


co o 


<j m 

o in 

i 

cr 

M 


mmm. 




IVAIAanS lN30d3d 


0t7-D£ 


02-D2 


Figure 21-7. Survival of Megalopa and Early Juvenile Crabs of 
Callinectes Sapidus Maintained at Three Cycles of Temperature 
(20-25°C, 25-30°C, and 30-35°C) r Three Salinities 
(15 ppt, 25 ppt, and 35 ppt) when Exposed to Two Dilutions 

of Methoprene. 


330 






























































































CAL LINECTES SAPIDUS 
ZR- SIS* CYCLIC TEMPERATURE 



*1 fll* 



S ■ # 


■S _ O _ 4§- 


o*« 


•B- 


C 01 ppm 

30-35*C 



0 »■§ 



a 



01 ppm 


Figure 21-8. Time Required for Metamorphosis of Megalopa of 
Callinectes Sapidus (M-1C) and Intermolt Periods of Subsequent 
Juvenile Stages (1C-2C, 2C-3C, and 3C-4C) when Maintained at 
Three Cycles of Temperature (20-25°C, 25-30°C, and 30-35°C), 
Three Salinities (15 ppt, 25 ppt, and 35 ppt) when Exposed to 

Two Dilutions of Methoprene. 

NOTE: The vertical column represents the range and the horizontal line the 
mean. 


331 


































































megalop stage was reduced in concentrations ot 0.01 ppm and 0.0001 
methoprene, but there was no significant effect on the survival lor larvae 
maintained in higher salinities. The developmental time was not significantly 
altered by the two lower concentrations of the compound and super-numerary 
larval stages were not observed in any of the experimental series. There was 
evidence that the early megalopa stage represented a period of extreme 
sensitivity to environmental stress in any form, including the presence of 0.1 
ppm methoprene when combined with 5 ppt or 35 ppt (Costlow, 1977). 

Christiansen, Costlow, and Monroe (1) reported a significant reduction in 
survival of zoeal larvae of Rhithropanopeus harrisii with increasing 
concentrations of methoprene (Altosid^: ZR-515) and further observed an 
increase in the duration of zoeal stages as the concentration of methoprene was 
increased, irrespective of changes in temperature or salinity. Below 0.1 ppm, 
methoprene did not inhibit metamorphosis. The work with a second 
compound, hydroprene (Altozar^: ZR-512) also resulted in a significant 
reduction of survival of larvae of Rhithropanopeus harrisii , and the first stage 

larvae appear to be the most sensitive stage within the four zoeae and one 
megalopa. Metamorphosis to the first crab stage was not inhibited at 
concentrations of 0.5 ppm or lower. 

An additional study on the way in which a third compound, MONO-585, 
affected the response of larvae of R. harrisii to light, indicated that both 
swimming speed and phototaxis were altered by the presence of this compound 
at sublethal concentrations (8). Further information on how this general group 
of compounds may affect a variety of physiological and developmental 
processes in marine crustacean larvae, however, is needed to determine if the 
effects observed by previous authors are limited to the relatively few 
compounds and few species which have heretofore been studied. 

From the present study it would appear that the compound MONO-585 is 
not as toxic as methoprene. Although at the concentration of 10 ppm, 
MONO-585 was lethal to larval stages of R. harrisii at salinities 5, 20 and 35 
ppt, 1 ppm of this compound was only lethal when it was combined with a 
salinity known to represent a stress condition to the developing larval stages (5 
ppt). In similar studies on the effect of methoprene on the development of R. 
harrisii (4) concentrations of 0.01 ppm and 0.0001 ppm methoprene resulted 
in a reduction in survival of larvae at a salinity of 5 ppt but did not 
significantly affect survival of larvae maintained in the higher salinities. A 
concentration of 0.1 ppm MONO-585 had no obvious effect on survival in any 
of the experimental salinities. As with experiments on methoprene, duration of 
the four zoeal stages and one megalopa of R. harrisii was not affected by the 
lower concentrations of MONO-585. 


332 


Previous studies on survival and length of life of megalopa of Callinectes 
sapidus (3) have indicated that they will withstand a wide range of salinity and 
temperature and display remarkably uniform survival in all but the lower 
salinities (5-10 ppt). In the present experiment, survival of the control series at 
20 and 35 ppt was similar to that recorded for previous studies, but the 
reduction in survival of the 10 ppm and 1 ppm MONO-585 clearly indicate the 
toxicity of this compound to the late larval stages of the commercial crab 
(Figure 21-3). Total mortality was observed when the larvae were exposed to 
10 ppm regardless of the salinity, and at 1 ppm, survival was reduced to 
approximately 40 percent, while survival at 0.1 ppm resulted in a slight 
reduction in survival relative to the control series (Figure 21-3). As with R. 
harrisii (4), there was no significant reduction in time required for 
metamorphosis, regardless of the concentration of MONO-585 (Figure 21-4). 

Earlier studies on the effect of cyclic temperatures, as opposed to constant 
temperatures, on the survival of larvae of the mud-crab Rhithropanopeus 
harrisii (6, 16) indicated that at one particular five degree cycle of temperature, 
30-35°C, a significantly higher survival could be expected relative to that 
observed in a constant temperature of either 30°C or 35°C. In the present 
study with megalopa of Callinectes sapidus , the only obvious effect on survival 
in three cycles of temperature combined with three salinities and two 
concentrations of MONO-585, was also associated with the high cycle of 
temperature, 30-35°C (Figure 21-5). Although there was no significant 
reduction in survival at 15 or 25 ppt combined with 1.0 ppm MONO-585, 
megalopa maintained in a salinity of 35 ppt, 1.0 ppm MONO-585, showed a 
significant reduction in survival at a cycle of 20-25°C and at 25-30°C. When 
the megalopa were maintained at 1.0 ppm MONO-585, 30-35°C, survival was 
considerably increased but, as with the study on larvae of R. harrisii , there is at 
present no obvious explanation as to how this high cycle of temperature 
contributes to an increase in survival of the larval stages. 

Very little information is available on the way in which early juvenile stages 
of any crab respond to natural environmental conditions or artificial 
compounds which may be present within the water. From the present study it 
would appear that the intermolt period for the first four juvenile crabs may 
exhibit considerable variability, but this variability cannot be attributed to 
either salinity, temperature, or insofar as this experiment is concenrned, the 
presence of sublethal concentrations of either MONO-585 or methoprene 
(Figures 21-5 and 21-7). 

A broad range of questions remains concerning the physiological response of 
many crustacean larvae and adults to the juvenile hormone mimics in insect 
growth regulators. Nothing is known as yet as to how these compounds may be 
incorporated within the animal, or the way in which they may further alter 


333 


behavioral or locomotory patterns. While the short-term effects on 
development of two species of Decapoda are described in this paper, nothing is 
known of the way in which long-term exposure to sublethal concentrations 
through a number of successive generations may contribute to mutagenic 
effects. Within the realm of the chemistry of these compounds, a number of 
questions also remain unanswered. Several authors have described the rate at 
which insect growth regulators degenerate within certain natural and artificial 
environments (16, 18) but none of these studies have investigated the 
degradation rate in either an estuarine or a marine environment. Most of the 
research which has been conducted thus far has concentrated on the effects of 
the intact compound, and no data appear to be available on either the 
breakdown products which may occur under estuarine conditions, their fate in 
the natural marine environment, or the way in which they may affect 
developmental processes of marine invertebrate animals. 

Although it is clear that the juvenile hormone mimics and insect growth 
regulators may offer great potential as replacements for many of the more 
persistent pesticides, it seems equally clear that a considerable amount of 
research remains to be done to assure their proper use within the estuarine and 
coastal environments. 

ACKNOWLEDGMENTS 


We are grateful to Zoecon Corporation, Palo Alto, California, for providing 
the pure compound methoprene (Altosid R ) and to the Monsanto Corporation, 
St. Louis, Missouri, for supplying MONO-585 for experimental use. This 
research was supported by a grant (R-803838-01-0) from the Environmental 
Protection Agency. 


REFERENCES 


1. Christiansen, M.E., J.D. Costlow, Jr. and R.J. Monroe. 1977a. Effects of 
the Juvenile Hormone Mimic ZR-515 (Altosid R ) on Larval Development 
of the Mud-Crab Rhithropanopeus harrisii in Various Salinities and Cyclic 
Temperatures. Mar. Biol., 39:269-279. 

2. Christiansen, M.E., J.D. Costlow, Jr. and R.J. Monroe. 1977b. Effects of 
the Juvenile Hormone Mimic ZR-512 (Altozar R ) on Larval Development of 
the Mud-Crab Rhithropanopeus harrisii at Various Cyclic Temperatures. 
Mar. Biol. 39:281-288. 

3. Costlow, J.D., Jr. 1967. The Effect of Salinity and Temperature on 
Survival and Metamorphosis of Megalops of the Blue Crab Callinectes 
sapidus. Helgolander Wiss. Meeresunters 15:84-97. 


334 


4. Costlow, J.D., Jr. 1977. The Effect of Juvenile Hormone Mimics on 
Development of the Mud Crab, Rhithropanopeus harrisii (Gould). Physiol. 
Responses Mar. Biota to Pollutants. Academic Press, pp. 439-457. 

5. Costlow, J.D., Jr. and C.G. Bookhout. 1959. The Larval Development of 
Callinectes sapidus Rathbun Reared in the Laboratory. Biol. Bull., 
116:373-396. 

6. Costlow, J.D., Jr. and C.G. Bookhout. 1971. The Effect of Cyclic 
Temperature on Larval Development in the Mud-Crab Rhithropanopeus 
harrisii. Proc. IV Eur. Sym. Mar. Biol. Cambridge University Press, pp. 
211 - 220 . 

7. Costlow, J.D., Jr., C.G. Bookhout and R.J. Monroe. 1966. Studies on the 
larval Development of the Crab, Rhithropanopeus harrisii (Gould). I. The 
Effect of Salinity and Temperature on Larval Development. Physiol. Zool., 
39:81-100. 

8. Forward, R.B., Jr. and J.D. Costlow, Jr. 1976. Crustacean larval Behavior 
as an Indicator of Sublethal Effects of an Insect Juvenile Hormone Mimic. 
In: Symposium on Behavior as a Measure of Sublethal Stress on Marine 
Organisms, Olla, B. (ed.), Third International Estuarine Research 
Federation Conference, Galveston, TX. New York: Academic Press, Inc., 
pp. 279-289. 

9. Gomez, E.D., D.J. Faulkner, W.A. Newman and C. Ireland. 1973. Juvenile 
Hormone Mimics: Effect on Cirriped Crustacean Metamorphosis. Science, 
N.Y., 179:813-814. 


10. Miura, T. and R.M. Takahashi. 1973. Insect Developmental Inhibitors. 3. 
Effects on Non-Target Aquatic Organisms. J. Econ. Ent., 66:917-922. 

11. Payen, G.G. and J.D. Costlow. 1977. Effects of a Juvenile Hormone Mimic 
on Male and Female Gametogenesis of the Mud-Crab, Rhithropanopeus 
harrisii (Gould) (Brachyura: Xanthidae). Biol. Bull., 152:199-208. 

12. Quistad, G.B., L.E. Staiger, and D.A. Schooley. 1975. Environmental 

Degradation of the Insect Growth Regulator Methoprene (isopropyl (2E, 
4E)-1 l-methoxy-3, 7, 11-trimethyl-2, 4-dodeca-dienoate). III. 

Photo-decomposition. J. Agr. Food Chem., 23:299-303. 

13. Ramenofsky, M., D. John Faulkner, and C. Ireland. 1974. Effect of 
Juvenile Hormone on Cirriped Metamorphosis. Biochem. Biophys. Res. 
Comm., 60:172-178. 


335 


14. Rosenberg, R. and J.D. Costlow. 1976. Synergistic Effects of Cadmium and 
Salinity Combined with Constant and Cycling Temperatures on the Larval 
Development of Two Estuarine Crab Species. Mar. Biol., 38: 291-303. 

15. Sacher, R.M. 1971. A Mosquito Larvicide with Favorable Environmental 
Properties. Mosquito News, 31:513-516. 

16. Schaefer, C.H. and E.F. Dupras, Jr. 1973. Insect Developmental Inhibitors. 
4. Persistence of ZR-515 in Water. J. Econ. Entomol., 66:923-925. 

17. Schaefer, C.H. and W.H. Wilder. 1972. Insect Developmental Inhibitors: A 
Practical Evaluation as Mosquito Control Agents. J. Econo. Ent. 
65:1066-1071. 

18. Schooley, D.A., B.J. Bargot, L.L. Dunham and J.B. Siddall. 1975. 
Environmental Degradation of the Insect Growth Regulator Methoprene 
(Isopropyl (2E, 4E) -1 l-methoxy-3, 7, 11-trimethyl-2, 4-dedecadienoate). 
II. Metabolism by Aquatic microorganisms. J. Agric. Fd. Chem., 
23:293-298. 

19. Slama, K., M. Romanuk and F. Sorm. 1974. Insect Hormones and 
Bioanalogues, 477 pp. Wien, New York: Springer-Verlag. 

20. Tighe-Ford, D.J. 1977. Effects of Juvenile Hormone Analogues on Larval 
Metamorphosis in the Barnacle Elminius modestus Darwin (Crustacea: 
Cirripedia). J. Exp. Mar. Biol. Ecol., 26:163-176. 


336 


STATISTICAL LINEAR MODELS IN THE 
COLLECTION AND ANALYSIS OF 
ECOLOGICAL DATA 


Saul B. Saila, Martin A.M. Hyman and Ernesto Lorda 

Marine Experiment Station 
Graduate School of Oceanography 
University of Rhode Island 
Kingston, Rhode Island 02881 


ABSTRACT 

The problems of environmental monitoring and baseline studies are 
considered. The importance of sampling is emphasized, and items to be 
considered are outlined. Time series analysis and linear models are discussed as 
two kinds of statistical methodology applied to environmental impact analysis 
and monitoring. Application of both these approaches is discussed and the 
reader is referred to comprehensive treatment in various references. While time 
series is mentioned only briefly, linear models are dealt with at length. The use 
of a simple linear model is illustrated by an example which relates to deciding 
where to establish monitoring stations along a cross-sectional area of a 
hypothetical estuary which is of interest. A scheme for collection of the data is 
presented along with the general analysis of variance table for this particular 
model. 

INTRODUCTION 

There seems to be no limit to the demands for. more and more data 
concerning the problems of prediction and protection of the marine and 
estuarine environments. From the volume of data being generated in some 
studies, the ultimate goal in monitoring and baseline establishment appears to 
be to measure everything, everywhere, continuously. It should be recognized 
that even if this virtually infinite amount of data were gathered, there is no 
guarantee that it would lead to complete understanding or predictive inferences 
from a given system. Thus, any environmental monitoring or baseline study 
should be practical and feasible within reasonable time and cost constraints. 
However, the data gathered must be accurate, pertinent to the problems at 
hand, concise and purposefully collected. 


337 


It is evident from the above that proper sampling of the marine environment 
is an important step in monitoring and impact assessment. For example, even 
though analytical methods for estimating certain environmental parameters 
may be highly accurate and precise, if the sample being analyzed is not 
representative, the data resulting from the analysis is relatively worthless. 

A sampling program for any environmental monitoring or baseline study 
must consider explicitly the following items: a) the number of samples 
required; b) sampling frequency; c) parameters to be measured; and 
d) sampling locations. These items are premised on some accepted definition of 
the level of perturbation or impact which is ecologically significant. It is 
recognized that a complete environmental assessment program encompasses a 
relatively comprehensive characterization (physical, chemical, biological) of a 
system, and includes determination of the potential impacts of pollutants or 
environmental changes on human health and ecological systems. Lucas (8) and 
Eberhart (5) have provided a review of some of the difficulties in assessing 
impacts and have proposed some models as bases for taking and analyzing 
environmental data. It is our belief that programs with more limited objectives 
of characterizing existing conditions or identifying previously defined impacts 
or changes can be developed as subsets of larger environmental assessment and 
monitoring programs. It is our objective to briefly describe some aspects of the 
design and analysis of such experiments to answer some specific questions on 
sampling with particular reference to ichthyoplankton. 

Most biotic elements of the environment are highly variable and 
everchanging. They must be sampled with sufficient intensity to determine the 
course of such changes in time and space. Empirical evidence to date 
concerning ichthyoplankton, as well as juvenile and adult fishes, suggests that 
several years of sampling may be necessary to detect reasonable changes in 
these populations. 

STATISTICAL METHODOLOGY 

There have been two general kinds of statistical methodology applied to 
environmental impact analysis and monitoring. They are linear model analysis 
and time series analysis. 

Time series analysis will not be considered in detail in this report. However, 
it will be briefly mentioned. In time-series analysis, the correlation of a 
response variable to past observations is taken into account in the formulation 
of a statistical model. Statistical time-series analysis has been treated in several 
excellent books including: Anderson (1), Box and Jenkins (2), and Nelson (9). 
The treatment of the methodology and time-series is comprehensive in these 
references, and the reader is referred to them for details. Our limited 


338 


understanding of the methodology suggests that relatively large amounts of 
data gathered at frequent and evenly spread sampling intervals are highly 
desirable for this methodology to be effective in most instances. 

The general linear model analysis is described at various levels of detail in 
several statistics books, such as Cochran and Cox (3), Davies (4), Federer (6), 
Kempthorne (7), Sheffe (10), and Snedecor and Cochran (11). This linear 
model approach includes analysis of variance and regression analysis. In this 
approach variations in a response variable measured over time and space are 
decomposed into assignable sources of variations and these variations are 
assumed to be additive. Tests of significance, such as F-tests or variance ratio 
tests to determine the change in the mean value of some variable from several 
sample events, are based on certain assumptions such as a normal probability 
density and independent and homogeneous variance (10). Data from samples 
taken over time frequently do not conform to these assumptions. 
Nonstationary elements, such as seasonal or diurnal, and tidal components are 
often present, and the data may be highly correlated in time. 

In the linear model approach time, space and sampling locations, along with 
replications, become a part of a planned experimental design. As a means for 
considering spatial and temporal variability in the linear model, the spatial and 
temporal distributions of biota (i.e., ichthyoplankton) are treated as a sum of 
responses due to assignable sources of factor levels. In addition, 
transformations of the response variable are sometimes used to achieve 
homogeneity of variances. Finally, because one can expect certain physical and 
biological data to be correlated, these relationships can be effectively utilized 
by carrying out multivariate analyses of variance and covariance analyses. 

In carrying out the linear model approach to monitoring and impact 
assessment, the method involves formulating hypotheses or linear contrasts for 
carrying out the statistical tests. Among these contrasts one tests for main 
effects due to a defined factor and the interaction of factors ot interest. 

SCHEME FOR ESTABLISHING SAMPLING STATIONS 

We will illustrate the use of a simple linear model by an example which 
relates to deciding where to establish monitoring stations along a 
cross-sectional area of an estuary which is of interest. For simplicity we will 
assume that cost constraints limit the number of samples to about 50. Our 
prior knowledge of the problem suggests that we should be concerned about 
the depth distribution of the given organism (say winter flounder larvae). Table 
22-1 gives a sketch of the data as they might be gathered for this analysis. No 
time effects are considered in this analysis. 


339 


Table 22-1. Numbers of Larvae Obtained by 
Sampling Different Depths Across an Estuary 


Stations on 

Cross Section 


Number of Larvae 


Surface 

Mid-depth 

Bottom 

la 

_ 

— 

— 

lb 

— 

— 

— 

1 c 

— 

— 

— 

2a 

— 

— 

— 

2b 

— 

— 

— 

2c 

— 

— 

— 

3a 

— 

— 

— 

3b 

— 

— 

— 

3c 

— 

— 

— 

4a 

— 

— 

— 

4b 

— 

— 

— 

4c 

— 

— 

— 

5a 

— 

— 

— 

5b 

— 

— 

— 

5c 

— 

— 

— 


The data in Table 22-1 illustrate an outline for triplicate determinations of a 
given ichthyoplankton larval species for five locations along the cross-section of 
an estuary at three depth levels. Since each station location has three replicate 
samples at each depth, the classifications “depths” and “locations” are 
completely crossed, while the triplicate determinations provide three 
replications for each combination of depth and location. In the above table 
there are three columns corresponding to depths, five rows corresponding to 
the five locations, and there are 15 cells each containing three replicate 
observations. 

In the general case there are xy a observations, i = l,...,p rows, j = l,...,q 
columns, a = 1,...,?? replicates. The model for the analysis is assumed to be: 

x ij a = M + I + ?)j + Xy + Sjj a • 

In the above model, ju is the mean, £ represents row effects (location 
variability), and r?j represents column effects (depth variability). The 
interaction effects Xy represent any variations which may be peculiar to a 
particular combination of station and depth, and the effects 5^ are normally 
distributed random components with average value zero for each ij. 


340 







The general analysis of variance table (Table 22-2) for this particular model 
is shown. From this table the partitioning of the degrees of freedom can be 
determined as well as the appropriate tests of significance determined. Two 
points can be made concerning this model. They are: 1) the variation between 
sample locations can be estimated, and 2) the systematic variations between 
sample locations can be eliminated from the study of other effects, i.e. depth. 

Assuming that an approach such as the above has been applied to 
establishing whether one or more sampling stations are required for the 
monitoring program, we now turn to a detailed analysis of a single station, 
which was a major component of the work which has been performed to date. 


341 




Table 22-2. General Analysis of Variance Table for First Stage Monitoring Station Selection 

(Basis for Table 22-1 Layout) 


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REFERENCES 


1. Anderson, T. W. 1971. The Statistical Analysis of Time Series. John Wiley 
and Sons, Inc., New York. 

2. Box, G. E. P. and G. M. Jenkins. 1970. Time Series Analysis, Forecasting 
and Control. Holden-Day, Inc. 

3. Cochran, W. G. and G. M. Cox. 1957. Experimental Design. John Wiley and 
Sons, Inc., New York, xiv + 617 pp. 

4. Davies, O. L. (ed.) 1954. Design and Analysis of Industrial Experiments. 
Oliver and Boyd, Edinburgh. 

5. Eberhardt, L. L. 1976. Quantitative Ecology of Impact Evaluation. J. 
Environmental Mgt. 4:27-70. 

6. Federer, W. T. 1955. Experimental Design Theory and Application. The 
MacMillan Co., New York. 

7. Kempthorne, O. 1952. The Design and Analysis of Experiments. 

8. Lucas, H. L. 1976. Some Statistical Aspects of Assessing Environmental 
impact. In: Proc. Conf. on the Biological Significance of Environmental 
Impacts, K. K. Sharma, J. D. Buffington, J. T. McFadden (eds.), 
NR-CONF-002. pp. 295-306. 

9. Nelson, C. R. 1973. Applied Time Series Analysis for Managerial 
Forecasting. Holden-Day, Inc., San Francisco. 

10. Sheffe, H. 1959. Analysis of Variance. John Wiley and Sons, Inc., New 
York. 

11. Snedecor, G. W. and W. G. Cochran. 1967. Statistical Methods 6th Edition. 
The Iowa State University Press, Ames, Iowa. 


343 


KANEOHE BAY: NUTRIENT MASS BALANCE, 
SEWAGE DIVERSION, AND 
ECOSYSTEM RESPONSES 


Stephen V. Smith 
Hawaii Institute of Marine Biology 
University of Hawaii 
P.O. Box 1346 
Kaneohe, Hawaii 96744 


ABSTRACT 

Kaneohe Bay, Hawaii, is a coral reef/estuary ecosystem presently subjected 
to stresses from sewage discharge and runoff. The sewage discharge is scheduled 
to be diverted from the bay. This “relaxation” of sewage stress will be a major 
ecosystem perturbation: the termination of a chronic stress which has been 
imposed, with increasing intensity, on the bay over the past two decades. We 
are treating this sewage diversion event as a controlled experiment designed to 
ascertain ecosystem responses to such environmental perturbation. The 
experiment is being performed by means of time-series field monitoring, 
discrete field studies, and laboratory experiments. 

The stream runoff imposes short-term, catastrophic stress from fresh water 
and sediment influx. The sewage accounts for about 90 percent of the 
land-derived nutrient delivery to the bay, thus imposing an influence which 
stimulates biological activity. 

The sediments in the bay have been a major repository for nutrients 
discharged into the bay; nutrient release from the sediments has been, and will 
continue to be, a significant process affecting the ecosystem. When the sewage 
stress is relaxed, planktonic responses to that event will be more rapid than 
benthic responses; both because the plankton are immediately responsive to 
the point-source sewage discharge, and because of characteristic high biomass, 
efficient nutrient cycling, and limited mobility of benthic organisms. 

INTRODUCTION 

Kaneohe Bay is a coral reef and estuary complex on the northeast 
(windward) coast of Oahu, Hawaii (Figure 23-1). The bay was once renowned 
as one of the most beautiful coral reef ecosystems in Hawaii. The reef 


344 



Figure 23-1. Index Map of Kaneohe Bay, Oahu, Hawaii, 
Showing the Locations of the Three Sectors of the Bay. 


community has deteriorated, and the waters have become more turbid in 
response to human perturbations. Chief among these perturbations have been 
domestic sewage discharge and stream runoff. Both processes have been closely 
related to the tenfold increase of the human population in the watershed over 
the past three decades. Banner (2) and Smith (8) have summarized the 
historical conditions leading to the present environmental status of the bay. 

The present stress regime is about to be drastically modified by diversion of 
the sewage discharge to a site removed from Kaneohe Bay. This paper 
discusses, from the bias of my own mass-balance approach to ecosystem 
analysis, interim results of a team investigation designed to ascertain ecosystem 
responses of Kaneohe Bay to the relaxation of sewage stress, and to derive 
predictive ability therefrom. The data, and many of the ideas presented here, 
are properly credited to other members of the research teamJ 


1. Working group leaders are: S. V. Smith (chemistry), E. Laws 
(phytoplankton), J. Hirota (zooplankton), R. E. Brock (benthos), P. L. 
Jokiel (microcosms). Principal cooperators from the Naval Ocean Systems 
Center are E. C. Evans III, J. G. Grovhoug, and R. S. Henderson. 


345 










The investigation combines field monitoring with field, microcosm, and 
laboratory experiments. The spatial distribution of variables in the bay is 
relatively well established; we have been gathering time-series data in the bay 
since early 1976. The outfall is due to be diverted shortly after this is written 
(November 1977), and we anticipate continued collection of time-series data 
for at least one year after the diversion. 


PROJECT DESIGN 

Kaneohe Bay is relatively well-described spatially, and methods by which 
chemical and biological characteristics of marine environments are measured 
are reasonably standardized. Therefore the analytical details of the study do 
not need discussion at this juncture. Let us instead examine the conceptual 
approach to this analysis. 

Sewage discharge presently imposes a large and well-documented loading of 
biologically active materials on the ecosystem. The change of that discharge 
volume with time is known, and the termination date of the discharge will be a 
discrete, well-defined event. The discharged materials alter the water 
composition near the discharge sites, become incorporated into the food web 
cycle within the ecosystem, and flush from the ecosystem. In addition to 
biostimulatory responses from the fertilization of the ecosystem, there may be 
responses from the loading of plant and/or animal toxins on the system. When 
the discharge terminates, there will be ecosystem responses as both direct and 
indirect ramifications of the sewage diversion. 

There are three main components to the present study. 

1. Routine field sampling, to document the sequence of chemical, plant, 
and animal changes through time. This sequence may be divided into “before 
diversion” and “after diversion” periods which may be compared as two 
distinct statistical populations of data, each of which may show seasonal or 
other temporal variations. The frequency of the time-series sampling is largely 
dependent upon the assumed or documented time scale of variability. For 
example, some water composition variables are measured one or more times 
per week, while characteristics of the benthos are measured every two months. 
Important adjuncts to the routine monitoring are the utilization of available 
Kaneohe Bay field data antecedent to our own, and use of data from 
environments which may be comparable to Kaneohe Bay with respect to some 
(but not all) of the natural and artificial ecosystem characteristics. 

2. Field studies, designed to answer specific questions about the ecosystem. 
These studies may also establish time sequences and spatial variations in the 


346 


bay, but they are not undertaken as ongoing routine monitoring. These 
measurements are being made before diversion and, to the extent necessary, 
will be repeated after the diversion. The sampling design is modified to answer 
specific questions. For example: What are the relationships among variables 
obviously related to water clarity? While an answer can, to some extent, be 
extracted from routine sampling, it is more satisfactorily addressed by sampling 
along strong water clarity gradients which may or may not coincide with the 
routine sampling stations. 

3. Laboratory experiments, also designed to answer specific questions 
about the ecosystem. Particular responses of communities within Kaneohe Bay 
are best addressed by controlled laboratory experiments. These experiments 
vary in volumetric scale from batch phytoplankton cultures in 500 ml flasks, to 
flow-through microcosm tanks which are 500 liters or larger in volume. The 
questions addressed in these simplified, but controlled laboratory experiments, 
cannot be easily answered under natural, and largely uncontrolled field 
conditions. Of course, the largest of the controlled experiments is the bay 
itself, a “reaction vessel” with a water volume in excess of 200 million m^. The 
time scales of these experiments vary from a few days in the flasks, to months 
in the microcosms, and several years in the field. 

In this presentation, I do not explicitly separate these various research 
components. Rather, I synthesize the components into our present view of 
total ecosystem characteristics and predicted responses to sewage diversion. 
This exercise is, of necessity, a preliminary analysis of our ongoing study. 

MAJOR ECOSYSTEM CHANGES IN THE 
PAST TWO DECADES 

The impact of runoff on Kaneohe Bay is largely in the form of short-term 
“catastrophic events.” In the past 17 years, there have been three years with 
monthly rainfall in excess of 75 centimeters within the Kaneohe watershed 
(Figure 23-2). In terms of water delivery to the bay, May 1965 represented an 
extreme: most of the rain fell in a 2-day period and was followed by rapid 
runoff. A freshwater lens from that storm killed corals and other reef 
organisms on the fringing reef and nearshore patch reefs to a depth of up to 1.5 
meters (1). The reef flats are less than 1 meter deep, so such a destructive 
“freshwater kill” virtually decimated the stenohaline marine organisms of the 
reef flats and upper portion of the reef slopes. Below about 2 meters the 
organisms were relatively unaffected. 

Sediment loading associated with runoff has two general effects on the 
ecosystem, one as the material is deposited, the other as the material is in the 
water column. Deposition smothers reef organisms and lowers the availability 


347 


H 

< 

H 

Q_ 

O 

LlI 

cr 

CL 


900 - 


800 


700 


600 


500 


400 


300 


200 


100 


KANEOHE MAUKA 
STATION 



60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 

YEARS 

Figure 23-2. Monthly Rainfall Since 1960 at a Selected Rain 
Gauge in the Kaneohe Bay Watershed. 


of hard substratum for settlement; particulate material in the water column 
lowers light and interferes with feeding mechanisms. 

Nutrient loading from sewage discharge has increased about sixfold since 

1963 (Figure 23-3). This increase is consistent with the previously cited rate of 
human population increase in the Kaneohe watershed. Virtually the entire 
nutrient load delivered to Kaneohe Bay is stripped from the water by biological 
uptake. There have been several obvious responses to the increased nutrient 
loading. Benthic algae are locally abundant on the reef flats and compete 
successfully with the corals for space on the reef slopes (2). The zone of 
present algal dominance on the reef slopes corresponds with the zone left 
undamaged by the 1965 freshwater kill. Phytoplankton standing crop and 
productivity are elevated above pre-loading levels; included in this high 
standing crop are frequent plankton “blooms” (4, plus our own data). Various 


348 











































SEWAGE DISCHARGE (I0 3 m 3 /day) 



YEARS 

Figure 23-3. Volume of sewage discharge into Kaneohe Bay since 

1960. 


NOTE: The Kaneohe and Marine Corps discharge enters the southeast sector, 
whereas the Ahuimanu discharge flows into a stream which drains into the 
northwest sector. 


349 







detritivores are favored directly by organic loading associated with the sewage 
input, and indirectly by the products of inorganic nutrient loading (3, plus 
additional project data). 

The relative impact of streams, rainfall, and sewage on freshwater and 
nutrient delivery on Kaneohe Bay can be established by a simple budgetary 
analysis. Sewage contributes a minimal amount of freshwater (Table 23-1); 
however, sewage accounts for most of the nitrogen and phosphorus delivery 
(Table 23-2). 

The bay may thus be seen to be sporadically perturbed by fresh water and 
associated sediment delivery, and chronically perturbed by nutrient delivery. 
This latter perturbation, which has increased dramatically over the past two 
decades, will be terminated. 


Table 23-1. Spatial Distribution of Water Inputs to Kaneohe Bay 

(millions of m^/month) 

Process 


Runoff + Rain- 


Sector 

groundwater 

evap. 

Sewage 

Total 

Southeast 

2.6 

-0.7 

0.5 

2.4 

Central 

1.0 

-0.8 

0.0 

0.2 

Northwest 

5.0 

-1.6 

0.0 

3.4 

Total 

8.6 

-3.1 

0.5 

6.0 


Table 23-2. Total Loading of "New" Nutrients on Southeast 
Sector (thousands of moles/day) 

Process 


Nutrient_Sewage Streams Total 


Nitrogen 30.5 4.2 34.7 

Phosphorus 3.4 0.4 3.8 


350 







NUTRIENT FLUXES 


Figures 23-4 and 23-5 illustrate data for several water quality variables at 
three diverse locations in Kaneohe Bay. “OF” is within about 100 meters of 
the major sewer outfall; “S” is in the southeast sector of Kaneohe Bay; and 
C is in the central sector. Samples have also been taken from the northwest 
sector, where values are similar to those of the central sector. 

In order for net exchange of material to occur between two water masses, a 
concentration gradient of that material is required. An estimate of the net mass 
flux between two well-mixed source waters may be obtained as the exchange 
volume times the concentration difference between those water masses. The 
concentration differences illustrated by Figure 23-4 qualitatively demonstrate 



OF 

S 


dissolved 
organic N 




o.o 5.0 io.o 


ALL UNITS ARE mmole/m^ 


Figure 23-4. Concentration of Selected Water Quality Variables 
Near the Kaneohe Outfall (OF), Near the Middle of the 
Southeast Sector (S), and Near the Middle of the Central 

Sector (C). 

NOTE: The bars represent the mean values ± one standard error unit. 


351 


















OF 

S 

c 



0 




PN 


IOO 


OF 

s 

c 





POC 


1 -J 

250 500 


OF 

S 

C 



chlorophyll a 

_i_—i 

2 4 


OF h 
S 

C 


0 


microzooplankton 
dry weight 


250 


500 


OF 



s 



c 


0 


macrozooplankton 
dry weight 

Too 200 

ALL UNITS ARE mg/m^ 


Figure 23-5. Concentration of Selected Water Quality Variables. 


NOTE: Symbols explained on Figure 23-4. 


the following points: (1) there is net phosphorus dispersal from the outfall to 
the southeast sector, and from the southeast sector to the central sector; and 
(2) nitrogen flux occurs from the outfall to the southeast sector, but 
insignificant from there to the central sector; and (3) there are also fluxes of 
the various particulate materials (Figure 23-5). 

Quantitatively, just how significant are these fluxes? The mean residence 
time of water in the southeast sector of Kaneohe Bay has been variously 
estimated but is apparently about 20 days (10). The volume of the southeast 
sector is about 80 x 10^ m^, so the daily exchange volume is about 4x10^ 
m^/day. Table 23-3 summarizes nitrogen and phosphorus fluxes calculated 
from the concentration gradients and exchange volume, and compares these 
fluxes with input rates of “new” nitrogen and phosphorus. There is a dramatic 
imbalance between stream plus sewage input to the southeast sector, and 
advective output from that sector. The calculation is not entirely accurate, 
because some material is known to pulse from the southeast sector in a 


352 
















Table 23-3. New Nutrient Input Versus Oceanic Advection 
from the Southeast Sector (+ is in) (thousands of moles/day) 

Process 


Nutrient 

Sewage 

Streams 

Advection 

Imbalance 

Fixed inorg. N 

+ 15.8 

+2.0 

-0.6 


Dissolved org. N 

+13.5 

+1.0 

-3.6 


Particulate N 

+ 1.2 

+1.2 

-4.5 


Total N 

+30.5 

+4.2 

-8.7 

+26.0 

Inorg. P 

+ 2.6 

+0.2 

-1.0 


Dissolved org. P 

+ 0.7 

+0.1 

i 

o 

CJ 


Particulate P 

+ 0.1 

+.01 

-0.5 


Total P 

+ 3.4 

+0.4 

-1.8 

+ 2.0 


low-density plume which flows northwestward from the sewer outfall. 
Nevertheless, either this simple mixing model underestimates advective losses 
by two-three fold, or there are additional budgetary terms to be considered. 

The budget can be further amplified. There is uptake of nutrients by 
planktonic and benthic algae, and subsequent cycling of these particulate 
materials within the food web. There is fallout of particulate organic material 
to the lagoon floor and nutrient release from the lagoon floor back into the 
water column. We have obtained nutrient release rates, gathered over one year 
by using 1-meter diameter Plexiglas hemispheres as in situ incubation 
chambers, and we can solve for fallout by difference between nutrient inputs 
and outputs (Table 23-4). The advective flux of nutrients from the southeast 
sector equals 30-50 percent of the nutrient inputs from terrigenous sources. 
The fallout of particulate nitrogen substantially exceeds terrigenous nitrogen 
inputs to the southeast sector. The high nitrogen fallout is maintained by rapid 
nitrogen release from the sediments. Particulate phosphorus fallout is also high, 
although it does not quite exceed terrigenous inputs. As with nitrogen, the 
rapid phosphorus fallout is maintained in large part by nutrient release from 
the sediments. The steps from stream plus sewage input to particulate fallout 
to release from the sediments show a progressive increase in the N:P ratio 
(9-*14->18). Material advected from the southeast sector is proportionally low 
in nitrogen (N:P « 5), largely reflecting the virtually complete uptake of 
dissolved inorganic nitrogen from the water. 


353 






Table 23-4. Total Nutrient Budget for the Southeast Sector 
(+ is in) (thousands of moles/day) 

Process 


Sediment 

Nutrient Sewage Streams Advection Release Fallout 


Fixed inorg. N 

+15.8 

+2.0 

-0.6 

+14.4 

0.0 

Dissolved org. N 

+13.5 

+1.0 

-3.6 

0.0(3) 

0.0 

Particulate N 

+ 1.2(1) 

+1.2(2) 

-4.5 

0.0(4) 

-40.4 

Total N 

+30.5 

+4.2 

-8.7 

+14.4 

-40.4 

Inorg. P 

+ 2.5 

+0.2 

-1.0 

+ 0.8 

0.0 

Dissolved org. P 

+ 0.7 

+0.1 

-0.3 

0.0 

0.0 

Particulate P 

+ 0.1 

+0.1 

-0.5(5) 

0.9 

- 2.8 

Total P 

+ 3.4 

+0.4 

-1.8 

+ 0.8 

- 2.8 


(1) Particulate N & P calculated as follows. Steinhilper (9) gives sewage Part. 
N « 1 g/m^; flow is 18 x 1(P m^/day; assume N:P= 10:1. 

(2) Particulate N & P in streams from stream carbon by Steinhilper (9), plus 
assumption of C:N:P= 100:1 5:1.5. 

(3) Our limited data plus Hartwig's (5) data show dissolved org. N and P 
flux from sediment is small. 

(4) Sediment resuspension of organic material excluded from calculation. 

(5) Assume particulate N:P= 10:1. 


The lagoon sediments are the repository for most of the “new,” or 
terrigenous, nutrients which have been delivered to the southeast sector of 
Kaneohe Bay. There is a substantial cycling of nutrients between that 
repository and the water column, with surprisingly little loss (especially of 
nitrogen). This situation has been recognized on the basis of a water-column 
nutrient budget. We do not yet have enough sediment nutrient data to establish 
a quantitatively defensible sediment nutrient budget, but the nutrient level in 
the sediment lends qualitative support to the assertion. 


354 





PREDICTED BIOLOGICAL RESPONSES TO SEWAGE 
DIVERSION 


The post-sewage diversion delivery of land-derived nutrients to the bay will 
decrease to about 10 percent of the present delivery (Table 23-2). The 
sediment reservoir will temporarily continue to release nutrients, but that 
reservoir must eventually be depleted as a fraction of the released material is 
constantly lost to advection. The sediment nutrient release to the water 
column is diffuse; while that release is sufficient to sustain a high total standing 
crop of plankton, that standing crop will not be as locally concentrated as the 
crop presently sustained by the point-source sewage input (Figure 23-5, 
chlorophyll). 

The central and northwest sectors of the bay presently has *^*c productivity 
rates of about 5 mg c m'^ hr'*, in comparison with about 9 mg C m° hr'* in 
the southeast sector and 24 mg C m'^ hr'* near the sewer outfall. There is 
relatively little inorganic nutrient export from the southeast sector to the other 
sectors (Table 23-4), so those sectors are not directly affected by the sewage. 
They are indirectly affected, because particulate material produced from the 
sewage nutrients is swept from the southeast sector and is sedimented in the 
other sectors, where it then releases nutrients back to the water column. 
Without the sewage point-source “new” nutrient input to the southeast sector, 
phytoplankton productivity there will stabilize near that of the other sectors. 
Except in the immediate vicinity of the present sewage plume, actual 
planktonic biomass decrease associated with the diversion should be small. 
Compositional shifts of both phytoplankton and zooplankton will 
undoubtedly occur, but we do not anticipate a significant change in the 
number of species present. We do anticipate a decrease in the abundance of 
certain meroplankton (e.g., barnacle larvae). 

The benthos will also respond to sewage diversion, but more slowly than the 
plankton. The relatively large biomass, longevity, and relative immobility of 
the benthic organisms provide a substantial nutrient pool which is not as 
efficiently removed from the system as are suspended and dissolved materials. 
Some of the filter-feeding benthic animals, particularly those immediately 
within the sewage plume, will not survive lowered food availability. Biomass 
may gradually drop, but plant-animal symbioses and other relatively “tight” 
pathways of nutrient cycling within the benthos community are mutualistic 
strategies which will tend to preserve the status quo. Efficient internal cycling 
of phosphorus has been demonstrated in shallow reef benthos communities 
elsewhere (6,7). Nitrogen is not as efficiently retained as phosphorus (11); 
however, experiments we have performed suggest that, given adequate 
phosphorus reserves, the reef benthos community can rely on nitrogen fixation 
and nitrification to supplement fixed nitrogen losses (see also 12, 13). 


355 


Eventually, discrete events will disrupt portions of the benthos community. 
Strong onshore winds rip the benthic algae loose from the bottom, and some of 
that material is swept from the system. The filter feeding animals and other 
detritivores will largely survive until they are killed by fresh water and/or 
sediment inputs, although some will starve from lowered planktonic food 
availability. 

The benthos community of the southeast sector is dramatically different 
from the reef community which was once found there, although historical data 
are insufficient to document the gain or loss of taxa. Corals, which once 
dominated the reefs there, as elsewhere in the bay, survive as isolated 
specimens. Benthic algal biomass locally exceeds pre-sewage biomass and shows 
large temporal fluctuation. The reef community structure has been obliterated. 
Benthos recovery will be back towards a coral-dominated, low-algal biomass 
community only if there is adequate substratum for coral settlement; if the 
periods between the interruptions by freshwater runoff are sufficient for 
community succession to proceed to the successful recruitment of corals; and 
if sediment nutrient release cannot maintain the high algal biomass. Banner (1) 
reported some coral recovery, in areas not otherwise significantly stressed, 
within three years of the 1965 “freshwater kill” previously mentioned. A 
return to coral dominance, if it ever occurs, will probably take one or more 
decades; shorter-term recovery patterns should indicate the direction of 
environmental rebound. 

SUMMARY 

1. The present biological structure of Kaneohe Bay may be related to the 
combination of catastrophic lethal events (runoff) and chronic biological 
stimulation (sewage discharge). 

2. The nutrient deposition as particulate materials in bay sediments and 
subsequent release from those sediments is an important and previously 
undocumented part of the internal nutrient cycle within the bay. This efficient 
cycle allows very little nutrient loss from the bay and comprises an 
instantaneous nutrient delivery to the water column comparable in magnitude 
to the sewage input. Of course, the sediment release contrasts with the sewage 
input in being a diffuse, rather than a point-source, delivery of nutrients to the 
water column. 

3. The planktonic portion of the biota can respond rapidly to alteration of 
environmental regimes, by virtue of advective exchange with more nearly 
oligotrophic waters in the absence of the point-source sewage discharge. The 
plankton of the bay retain relatively minor vestiges of the 1965 freshwater kill. 
The plankton of the southeast sector should shift rapidly to a post-sewer 


356 


composition and activity. Some qualitative aspects of the plankton community 
— namely the composition of the meroplankton — will have longer term 
residual characteristics. 

4. As key components of the benthos change, their planktonic larvae 
should do likewise (e.g., barnacle nauplii, which dominate some plankton 
tows). The style of benthos succession after the 1965 freshwater kill has been 
influenced by sewage loading, towards a high plant and animal biomass, 
filter-feeding, and detritivore community. There will be a lag in the benthos 
response to sewage diversion. The lag will last until catastrophic events disrupt 
the long-term inertia maintained by the high biomass, limited mobility, and 
mutualism of material cycling among the benthic organisms. 

5. This relatively simple examination of mass balance, hydrography, and 
trophic structure provides a useful basis for predicting responses of the 
Kaneohe Bay ecosystem to sewage diversion. As we test the predictions by 
post-diversion observations and continued experiments, we will be able to 
refine and generalize our predictive ability further. 


ACKNOWLEDGEMENTS 

This study is funded by U. S. Environmental Protection Agency grant 
R803983 and by the Hawaii Marine Affairs Coordinator. The Sewers Division 
of the City and County of Honolulu has provided information for the study. 
The investigation is being undertaken by the Hawaii Institute of Marine 
Biology in cooperation with the Naval Ocean Systems Center. I thank the 
working group leaders and other investigators for this cooperation in this team 
endeavor. Hawaii Institute of Marine Biology Contribution Number 533. 

REFERENCES 

1. Banner, A. H. 1968. A Freshwater “Kill” on the Coral Reefs of Hawaii. 
Hawaii Inst. Mar. Biol. Tech. Rep. 75:1-29. 

2. Banner, A. H. 1974. Kaneohe Bay, Hawaii: Urban Pollution and a Coral 
Reef Ecosystem. In: Proc. 2nd Int. Coral Reef Symp. (Brisbane) 

2:685-702. 

3. Brock, R. E., and J. H. Brock, (in press). A Method for Quantitatively 
Assessing the Infaunal Community Residing in Coral Rock. Limnol. 

Oceanogr. 


357 



4. Caperon, J., S. A. Cattell, and G. S. Krasnick. 1971. Phytoplankton 
Kinetics in a Subtropical Estuary: Eutrophication. Limnol. Oceanogr. 
16 : 599 - 601 . 

5 . Hartwig, E. 0. (in press). The Impact of Nitrogen and Phosphorus Release 
from a Siliceous Sediment on the Overlying Water. Proc. 3rd Int. Est. Res. 
Conf. (Oct. 7-9, 1975) Galveston. 

6. Pilson, M. E. Q., and S. B. Betzer. 1973. Phosphorus Flux Across a Coral 
Reef. Ecol. 54:581-588. 

7. Pomeroy, L. R., M. E. Q. Pilson, and W. J. Wiebe. 1974. Tracer Studies of 
the Exchange of Phosphorus Between Reef Water and Organisms on the 
Windward Reef of Eniwetok Atoll. In: Proc. 2nd Int. Coral Reef Symp. 
(Brisbane) 2:87-96. 

8. Smith, S. V. 1977. Kaneohe Bay: A Preliminary Report on the Responses 
of a Coral Reef/Estuary Ecosystem to Relaxation of Sewage Stress. In: 
Proc. 3rd Int. Coral Reef Symp. (Miami) 2:578-583. 

9. Steinhilper, F. A. 1970. Particulate Organic Matter in Kaneohe Bay, Oahu, 
Hawaii. Hawaii Inst. Mar. Biol. Tech. Rep. 22:1-53. 

10. Sunn, Low, Tom, and Hara, Inc. 1976. Kaneohe Bay Data Evaluation 
Study. Report Prepared for the U. S. Army Corps of Engineers. 7 
separately Numbered Chapters Plus Appendices. 

11. Webb, K. L., W. D. Dupaul, W. J. Wiebe, W. Sottile, and R. E. Johannes. 
1975. Enewetak (Eniwetok) Atoll: Aspects of the Nitrogen Cycle on a 
Coral Reef. Limnol. Oceanog. 20 : 198-210. 

12. Webb, K. L., and W. J. Wiebe. 1975. Nitrification of a Coral Reef. Can. J. 
Microbiol. 27:1427-1431. 

13. Wiebe, W. J., R. E. Johannes, and K. L. Webb. 1975. Nitrogen Fixation in a 
Coral Reef Community. Science 188 : 251 - 259 . 


358 


REPLICABILITY OF MERL MICROCOSMS: 
INITIAL OBSERVATIONS 


Michael E. Q. Pilson, 

Candace A. Oviatt, Gabriel A. Vargo and Sandra L. Vargo 
Graduate School of Oceanography 
University of Rhode Island 
Kingston, Rhode Island 02881 


ABSTRACT 


Nine microcosms at the Marine Ecosystems Research Laboratory (MERL) 
were run in replicate during the fall of 1976. Each microcosm tank is 5.5 m 
high and 1 .8 m in diameter, contains 13 m of water and 0.8 itt of sediment, 
and sits outdoors exposed to ambient light. Water and sediment were from 
Narragansett Bay. Water from the bay was run through the tanks at a rate of 
330 ml per minute, resulting in a turnover time of about 27 days. 


In this paper a set of the data collected during the first four months of 
operation is examined to discover the extent to which the microcosms 
replicated or diverged from each other and from the bay. Total chlorophyll a , 
nutrients, counts of individual phytoplankton specifies, and some other 
observations show that while there was considerable variability among the 
tanks at any given time, their overall behavior in the major features of bloom 
dynamics and species succession was consistent with that observed in the field. 

INTRODUCTION 

The development of marine microcosms has accelerated in recent years, 
due to an increased interest in investigating the properties of complex 
ecological systems, in understanding the effects of pollutants or other 
pertubations on such systems, and in using microcosms to carry out 
biogeochemical experiments (13) (3) (8) (17) (16) (10) (9) (14). The 
imposition of artificial boundaries and the limitation in size inevitably cause 
microcosms to differ from the natural systems they model. Nevertheless, the 
need to carry out controlled experiments on systems which represent a higher 
level of organization than cultures of single species has encouraged various 
attempts to pursue microcosm research. 


359 


At present there are no accepted criteria by which it is possible to establish 
whether a microcosm behaves in a way similar to the natural system it is 
designed to mimic, or to judge whether its behaviour is satisfactory for use as 
an experimental tool. A major concern should be with replicability, but one 
difficulty here is that nature herself is highly variable, and it is not easy to 
properly frame the tests to be applied. 

In general, we suggest that if the enclosed ecosystems maintain similar 
species composition and diversity, if the metabolic rates in the systems and the 
major chemical fluxes and transformations are within the range of variability of 
the natural systems, and if the statistical behaviour of the systems is similar to 
that of the natural system, then it is reasonable to conclude that the major 
biological activities are carried on in similar ways. If so, one may have some 
confidence that the enclosed ecosystems are useful experimental tools. 

In this paper we analyse a portion of the data obtained during the first four 
months of running the microcosms at the Marine Ecosystems Research 
Laboratory, to examine their replicability with respect to each other and to 
Narragansett Bay. 

FACILITY AND PROCEDURES 
Narragansett Bay 

Since the microcosms to be described were designed in part to act as a 
model of Narragansett Bay, a brief introductory description of this bay is 
presented here. 

Narragansett Bay is about 40 km long by 18 km wide, oriented N-S with 
the mouth opening into Rhode Island Sound (Figure 24-1). The presence of 
islands causes a complex tidal current regime and some isolation of regions. 
Small fresh water inputs result in a weak salinity gradient from the mouth (31 
o/oo) to the northern end (V20 o/oo). The water column is generally well 
mixed, although slight stratification occurs at times. The annual temperature 
range is from -1°C to about 25°C. Sediments are generally a mixture of silt and 
clay, although sand is found in some locations. Tidal currents resuspend 
flocculent bottom sediments in the bay, which has an average depth of about 8 
m. The turnover time of the bay, based on a hydraulic model (USACE 1959) 
and a numerical hydrodynamic model (6) (7) is about 30 days. 

Phytoplankton populations in Narragansett Bay are characterized by a 
winter-spring diatom bloom, followed by multiple blooms of flagellates, 
diatoms, and micro-flagellates in the summer. There is considerable 
year-to-year variation in the occurrence and timing of the various blooms. 


360 



Figure 24-1. A Map of Narragansett Bay Showing the Location of 
the 13 Stations Sampled During the 1972-73 Survey, the Benthic 

Station and the Location of MERL. 


361 














Zooplankton of the bay are dominated by two species of Acartia which 
switch dominance depending on season. They are generally present in greatest 
biomass in late spring. During summer they are heavily grazed by larval fish, 
menhaden and ctenophores. The benthos of the bay consists mostly of 
heterotrophic soft bottom communities with Mediomastis sp. and Nucula sp. 
dominating numerically. Several areas of the bay have communities dominated 
by amphipods; where coarser sediments occur, large bivalves such as Mercenaria 
mercenaria and Pitar morrhuana may provide the most biomass. 

A eutrophication gradient exists in Narragansett Bay due to sewage inputs 
from the Providence River (about 380,000 m^/day). However, the lower bay is 
relatively clean and the water quality excellent. Average primary productivity 
at one station in the Bay, mostly due to phytoplankton, has been estimated to 
be 308 g C/yr (4) of which 45 percent may be comsumed by the benthos (12). 

Narragansett Bay, as well as much of the marine coastal waters of the 
northeast coast of the United States, is characterized by ecosystems in which 
most of the photosynthesis is carried out by phytoplankton, but in which the 
benthos plays an important part in the total cycling of energy and nutrients. 
The microcosm tanks were designed to maintain ecosystems functioning in a 
similar manner. The stirrers were designed to direct turbulent energy onto the 
sediments, thus effecting a resuspension of flocculent material. The tanks are 
exposed to natural sunlight, and their temperature regime follows that of the 
bay within a few degrees. 

Description of microcosms 

A brief description of the facility was presented by Pilson et al. (1977). 
Twelve fiberglass tanks are set up outdoors on land adjacent to a laboratory 
building. Figure 24-2 and Table 24-1 provide information on the tanks and 
some physical characteristics of the systems. All piping to the tanks is PVC or 
fiberglass, and water is pumped from a pier 30 m offshore by a diaphragm 
pump that appears to be non-destructive to plankton. 

Sediment in the microcosms is held in fiberglass containers in the bottom 
of each tank. The containers were filled with sediment collected north of 
Conanicut Island (near “benthic station,” Figure 24-1). An attempt was made 
to place the sediment in the right orientation in the containers, but inevitably 
considerable mixing occurred. Nevertheless, the major features of the benthic 
community in the tanks were similar to those in the bay during the period of 
the experiment (F. Grassle, personal communication). 

Nine of the tanks were first filled during August, 1976, and maintained on 
a flow-through regime (330 ml/min) giving a turnover time of about 27 days. 


362 



Figure 24-2. Diagram on One of the MERL Tanks. 

NOTE: Each fiberglass tank is insulated and has three flanged ports on the side 
and one drainage port. The sediment container, also of fiberglass, contains 
about 30 cm of sediment. The tanks are filled through a port on the side and 
water exits from about 1 m below the surface through a level-control stand 
pipe. The depth of water is about 5 m. The mixer moves vertically through an 
excursion of about 60 cm with a frequency which is variable but is now set at 
about 5 cycles per minute. 


Table 24-1. Characteristics of MERL microcosms 


Tank interior diameter, 1.83 m 
Tank interior height, 5.49 m 
Surface area, water, 2.63 
Depth of water, 4.98 m 
Volume of water, net, 13.0 m 
Salinity, 30 o/oo 


Mass of water, 13.3 tons 

o 

Area sediment, 2.52 m 
Depth of sediment, 0.30 m 

3 

Volume of sediment, 0.756 m 
Mass of sediment (wet), 1.10 tons 
Mass of sediment (dry), 0.568 tons 


363 









































Except for some difficulties associated with the initial operations, the nine 
tanks were run identically during the first four months in order to assess the 
replicability of the systems. Some of the data obtained during this time are 
used in this paper to test the similarity of the tanks to each other and to 
Narragansett Bay. 

Biological measurements used in comparisons 

Table 24-2 gives a list of the data used in the analyses to be presented here. 
A brief description of the analytical procedures follows. 

Nutrient analyses were performed using Technicon AutoAnalyser 
procedures somewhat modified for our purposes. Chlorophyll a was 
determined by the fluorometric method of Holm-Hansen et al. (1965). 

Phytoplankton were counted on 1-ml aliquots from a pooled sample (3 
depths pooled) from each MERL tank or from single samples taken at the end 
of the dock or in the bay. Generally the samples were counted live using a 

Table 24-2. List of Measurements Yielding Data Used 
for Intercomparisons; Weekly Sampling During 
August-December 1976 Unless Noted 



MERL 

9 microcosms 

Narragansett Bay* 
a b c 

ammonia 

X 

X 

X 

— 

nitrate i nitrite 

X 

X 

X 

— 

silicate 

X 

X 

X 

— 

phosphate 

X 

X 

X 

— 

chlorophyll a 

X 

X 

X 

— 

phytoplankton species counts 

X 

X 

X 

X 

zooplankton biomass 

X 

X 

X 

X 


*Narragansett Bay 

a. Data from a year-long biweekly survey made in 1972-73 at stations 
(surface and bottom). 

b. Input water to MERL microcosms, sampled from end of dock near intake 
line, close in time to weekly sampling of the tanks. 

c. Three stations (S. Quonset, S. Patience Island, and Ohio Ledge area) 
sampled weekly from September to November 1976, from Durbin and 
Durbin (unpublished data). 


364 





Sedgwick-Rafter cell, but when this was not possible counts were done within a 
few days of collection on samples preserved with Lugol’s iodine. 

Zooplankton biomass was measured on split fractions of a pooled sample 
of two 1-m net tows from each tank. The sub-samples were rinsed with 
deionized water, lyophilized and weighed. 

The greatest part of the total data set consisted of phytoplankton counts. 
About 74 species or species categories were identified in the tanks (Table 
24-3), but those that appeared five times or less were eliminated from the 
correspondence analysis (2). The remaining 54 species are identified in Table 
24-3 by a number in brackets following the species name. Generally only 3 to 
20 species were found at one time in any individual count. 

RESULTS 

When the microcosms were started in mid-August, 1976, the 
phytoplankton concentration in the Bay was decreasing after a very dense 
bloom (Figure 24-3). Concentrations continued to decrease until the end of 
August, and thereafter stayed low until the middle of November when another 
bloom began. The MERL microcosms followed a similar course, with the 
second bloom beginning somewhat earlier in some of the tanks than in the Bay. 
The values for chlorophyll a in water samples taken from the end of the GSO 
dock in all cases fell within the range of values plotted for the MERL 
microcosms. 

Figure 24-4 shows a plot of the number of species of phytoplankton 
counted in samples from the MERL microcosms, from the end of the dock and 
from three other stations. In nearly every case the number of species in samples 
from the dock lies within the range of total species reported for the tanks. 
Occasionally the values for the other three stations in the Bay lie outside the 
range in the tanks, but the variation appears random and the data sets do not 
appear to be separable. 

Indices of diversity and similarity were calculated using the phytoplankton 
species counts for the period noted. The Shannon index of diversity (Pielou 
1969), which takes account of the abundance of each species, was calculated 
for each of the tanks and the dock for each of the weekly samples (Table 
24-4). The mean value for the dock samples was higher than for the tank 
samples, indicating a somewhat greater phytoplankton diversity in the adjacent 
bay than in the tanks. 


365 


Table 24-3. List of Phytoplankton Species or Categories 
to which the Counts in Samples from the MERL Tanks 

have been Assigned 


Diatoms 

1. Asterionella japonica (1) 

2. Attheya decora (2) 

3. Biddulphia aurita 

4. Ceratulina bergonii (3) 

5. Chaetoceros affinis (4) 

6. Chaetoceros compressus (5) 

7. Chaetoceros costatus 

8. Chaetoceros curvisetum (6) 

9. Chaetoceros danicus (7) 

10. Chaetoceros decipiens (8) 

11. Chaetoceros didymus 

12. Chaetoceros gracilis (9) 

13. Chaetoceros lorenzianus (10) 

14. Chaetoceros subtilus v. abnormis (11) 

15. Chaetoceros perpusillus (12) 

16. Chaetoceros sp. (solitary, small) (13) 

17. Coretheron hystrix (14) 

18. Coscinodiscus concinnus 

19. Coscinodiscus spp. (1 5) 

20. Coscinosira polychorda 

21. Dactyliosolen mediterraneus 

22. Detonula confervacea (16) 

23. Ditylum brightwelli (17) 

24. Eucampia zoodiacus (18) 

Flagellates 

49. Amphidinium spp. (38) 

50. Dinobryon spp. (39) 

51. Dinophysis sp. (40) 

52. Distephaneus speculum 

53. Ebriasp. (41) 

54. Eutreptia sp. 

55. Glenodinium sp. 

56. Exuviaella baltica 

57. Exuviaella sp. 

58. Gymnodinium simplex (42) 

59. Gymnodinium sp. 1 

60. Gymnodinium spp. (43) 

61. Gyrodinium sp. (44) 


25. Guinardia flaccida 

26. Lauderia borealis (19) 

27. Leptocylindrus danicus (20) 

28. Leptocylindrus minimus (21) 

29. Melosira nummoloides 

30. Nitzschia closterium (22) 

31. Nitzschia longissima (23) 

32. Nitzschia seriata (24) 

33. Phaeodactylum tricornutum (25) 

34. Rhizosolenia delicatula (26) 

35. Rhizosolenia fragilissima (27) 

36. Rhizosolenia hebetata 

37. Rhizosolenia setigera (28) 

38. Skeletonema costatum (29) 

39. Stephanopyxis turris 

40. Thalassionema nitzschioides (30) 

41. Thalassiosira aestivalis (31) 

42. Thalassiosira decipiens 

43. Thalassiosira nordenskioeldii (32) 

44. Thalassiosira rotula (33) 

45. Thalassiosira sp. (solitary cells) (34) 

46. Thalassiosira sp. (unidentified) (35) 

47. Thalassiothrix frauenfeldii (36) 

48. n pennates (37) 


62. Masartia rotundatum (45) 

63. Olisthodiscus luteus 

64. Oltmanziella sp. 

65. Peridinium steinii 

66. Peridinium triquetrum (46) 

67. Peridinium trochoideum (47) 

68. Peridinium spp. (48) 

69. Prasinocladus sp. (49) 

70. Prorocentrum redfieldii (50) 

71. Prorocentrum scutellum (51) 

72. Prorocentrum triangulatum (52) 

73. Pyramimonas torta (53) 

74. ju flagellates (54) 


366 


/ 





o 



Figure 24-3. Chlorophyll a Concentration in: a. the MERL 
Microcosms (Mean and Range) During the Replicability 
Experiments; b. Narragansett Bay at the End of the 
Graduate School of Oceanography Dock During 
the Same Period of Time. 


367 





















40 


CO 

UJ 

o 

UJ 

CL 

cn 30 


o 

h- 



0 - 1 - 1 - 1 - 1 — 

9/4 9/24 10/14 11/3 11/23 


Figure 24-4. Species of Phytoplankton 

NOTE: Solid lines indicate the range of the number of species of 
phytoplankton counted in samples from the MERL microcosms. The large 
opaque circles indicate the number of species from the dock samples. Three 
small dots connected by a vertical line indicate the number of phytoplankton 
species for three stations in upper Narragansett Bay (Durbin and Durbin, 
unpublished data). 


In order to assess the similarity in types of species in the community a 
similarity index (13) was calculated for each weekly data set. This index (S = 
2C/(A+B)) is a measure of the number of species in common between two 
samples. Calculations were made for each tank in comparison with the dock 
samples, and for some tanks in comparison with each other. Some of these 
indices are shown in Table 24-4. In general, the inter-tank similarity indices 
were about the same magnitude as the bay-tank indices. 


368 










Table 24-4. Comparisons of Phytoplankton in the Microcosms and in 
Narragansett Bay at the GSO Dock During 
the Replicability Experiment. 


NOTE: Similarity indices for the comparison of bay with all microcosms and 
for microcosm 3 with all other microcosms. Shannon index of diversity (H) for 
the microcosms and the bay. 





Similarity Index (S) 


Shannon Index (H) 



Bay-Tank 

Comparison 

Tank 3-Tanks 
Comparison 

Tank 

Bay 

Date 

Mean 

Range 

Mean 

Range 

Mean 


Aug. 

16 

0.379 

0.267-0.485 

0.608 

0.533-0.710 

0.263 

0.248 


30 

0.289 

0.143- 0.944 

0.617 

0.500-0.800 

0.021 

0.270 

Sept. 

13 

0.442 

0.267-0.640 

0.445 

0.261-0.593 

0.247 

0.270 


20 

0.325 

0.111-0.552 

0.398 

0.222-0.552 

0.327 

0.424 


27 

0.185 

0.074-0.333 

0.328 

0.231-0.581 

0.288 

0.093 

Oct. 

12 



0.373 

0.200-0.500 

0.171 

— 


18 

0.262 

0.133-0.429 

0.308 

0.125-0.571 

0.048 

0.520 


27 

0.286 

0.190-0.545 

0.458 

0.316-0.667 

0.209 

0.391 

Nov. 

8 

0.584 

0.421-0.696 

0.683 

0.452-0.824 

0.524 

0.695 


15 

0.642 

0.571-0.727 

0.612 

0.516-0.769 

0.441 

0.525 


22 

0.584 

0.457-0.667 

0.671 

0.545-0.824 

0.586 

0.795 


29 

0.639 

0.208-0.757 

0.678 

0.345-0.762 

0.471 

0.769 

Dec. 

6 

0.660 

0.467-0.788 

0.669 

0.588-0.757 

0.558 

0.810 


14 

0.549 

0.483-0.628 

0.584 

0.455-0.714 

0.592 

0.933 


22 

0.530 

0.414-0.648 

0.584 

0.455-0.709 

0.599 

0.807 


28 

0.620 

0.480-0.743 

0.536 

0.428-0.667 

0.607 

0.890 

Mean 


0.465 


0.535 


0.372 

0.563 

Std. Dev. 

±0.163 


±0.130 


±0.201 

±0.270 


While we do not have concurrent data on nutrients and chlorophyll from 
more than one station in Narragansett Bay, the results of a year-long survey at 
13 stations in the bay taken in 1972-73 are available. Data from three of these 
in the lower west passage of the bay are shown in Figure 24-5. The data set 
from the bay is in most respects similar to that from the MERL microcosms. 
Chlorophyll concentrations indicated a bloom in November in both the tanks 
and the bay which did not occur in 1972. Ammonia concentrations in the 
microcosms tended to be higher than in the bay, but mean values generally fell 


369 







CHLOROPHYL-R RMMONIR N02+N03 PHOSPHRTE 

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370 


Figure 24-5. Data Collected from Surface (x) and Bottom Water ( ) at Three Stations in the Lower West 
Passage of Narragansett Bay, During a Survey Carried out in 1972-73, are Compared with Mean Values 
from the Nine MERL Microcosms (0) During August 17 to December 31, 1976. 








within the range of variability in the bay. Phosphate concentrations in the 
microcosms tended to be higher than in the bay during the latter part of 
September and October. Comparison between the chlorophyll a graphs in 
Figure 24-5 and in Figure 24-3 indicates that the timing of the phytoplankton 
blooms may have been different in the two years. Nevertheless, the overall 
behaviour of the tanks and the bay is difficult to distinguish. Figure 24-5 also 
gives some indication of the patchiness and other variability apparent in 
Narragansett Bay. 

The variability and scatter of the magnitudes of individual measurements 
both in the bay and in the tanks show that it is difficult to distinguish tank 
behaviour from bay behaviour by using comparisons of single variables. In 
addition, various of the single variables are correlated, making statistical 
analysis of single variables less rigorous. Accordingly, multivariate statistical 
comparisons were attempted. 

A stepwise discriminant analysis (11) was performed on a weekly data set 
from the microcosms and bay input water collected from August 17 to 
December 6, 1976, to observe the replicability of the microcosms. The first 
two axes of this analysis explained 84 percent of the variation in the data set 
(Figure 24-6). The first five variables in the order of their importance 
(nitrate-nitrite, phosphate, ammonia, silicate and zooplankton) accounted for 
99 percent of the variation explained in the first two axes (Table 24-5). 
Chlorophyll concentration explained so little of the variation (less than 1 
percent) that the analysis did not include it. Generally microcosms 1,5,6,7, and 
8 were more similar to each other while microcosms 2,3,4, and 9 and bay input 
water showed a greater individuality. If the microcosms were exactly similar, 
10 percent of each group would be classified into itself and each of the other 
nine groups. In fact, microcosm 3 classified 47 percent to itself, microcosm 9 
classified 53 percent to itself and bay classified 44 percent to itself, indicating 
that these microcosms and the bay had the most individuality (Table 24-5). 

The individuality of microcosms 3,9 and bay, as indicated mainly by 
differences in nutrient concentrations in the discriminant analysis, was not 
reflected in phytoplankton species as analyzed by correspondence analysis (2) 
(Figure 24-7). The nine microcosms were not distinguishably different in their 
species composition from August to December. Initially all microcosms and the 
bay were tightly clustered on the lower right hand side of Figure 24-7. A 
bloom in November was reflected in a greater variability in microcosm location 
and species location (left hand side of Figure 24-7), but at no time was there a 
characteristic species or species group which caused clearly different 
microcosm location. 


371 


2 


84% VARIATION EXPLAINED 


c\J 



X 

< 



3 


BAY 


-2 1_I_I_I_I_ 

-2-IOI2 

AXIS I 


Figure 24-6. A Stepwise Discriminant Analysis of Data on 
Concentrations of Zooplankton Biomass, Chlorophyll, Ammonia, 
Nitrate Plus Nitrite, Phosphate and Silicate in 9 MERL Microcosms 
and Bay Input Water from August 17 to December 6, 1976, 
was Used to Prepare this Plot of Centroids in Reduced Space. 

NOTE: Detailed information on the Analysis is presented in Table 24-5. 


A comparison of microcosm data and bay data allows us to determine 
whether the greater variability of some of the microcosms makes them truly 
different from the bay. A second discriminant analysis was preformed with the 
same microcosm data set (nutrients, phytoplankton, zooplankton), and a 
similar set from the 1972-73 bay survey at 13 stations around the bay for the 
same period of the year (Figure 24-1, Table 24-2). In Figure 24-8, the bay 
stations are found positioned in a roughly linear arrangement, parallel to the 
first axis, which corresponds approximately to the eutrophication gradient in 
Narragansett Bay. Stations at the clean mouth of the bay and in the deeper east 
passage are positioned to the right. Stations in the west passage to the upper 
bay are progressively positioned to the left with the Providence River on the 
extreme left. All the microcosms, including those which exhibit more divergent 
behaviour, are positioned in mid-bay locations. Microcosm 2 appears 


372 



Table 24-5. Stepwise Discriminant Analysis of the 9 MERL Tanks and 
Bay Input Water from August 17 to December 6, 1976 


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Percent of group cases correctly classified 27.3%. Variable not used: Chlorophyll. Total variation explained: 
99%. Variables: 1 — NOg-NOg (62% variation) 2 — PO4 (23% variation) 3 — NH^ (7% variation) 4 — SiO^ (6% 
variation) 5 — Zoop. (1% variation). 






30 


PHYTOPLANKTON '00' 
MERL TANKS 0 

BAY INPUT WATER • 

37% VARIATION EXPLAINED 


2 0 


' 31 * 
4 


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Figure 24-7. Correspondence Analysis on 49 Phytoplankton Species 
('00') in 9 MERL Microcosms (0) and Bay Input Water (•) from 
September 14, to December 6, 1976. 

NOTE: In some cases sample locations (microcosms) are closely adjacent and 
not all of the 10 samples per microcosm are plotted. All variable locations 
(phytoplankton species) are plotted as accurately as possible. Microcosm 
location and phytoplankton species location were closely adjacent on the lower 
right hand side of the figure until the first part of November when blooms 
occurred in the bay and in the microcosms. The arrows indicate divergence 
from non-bloom conditions to bloom conditions in November when variability 
in species composition and microcosm behavior became greater. 


374 





3 


85% VARIATION EXPLAINED 


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Figure 24-8. A Discriminant Analysis of Data from the 9 MERL 
Microcosms and the 13 Bay Stations Using the Variables: 
Chlorophyll, Zooplankton, Ammonia, Nitrate Plus Nitrite, 

Phosphate and Silicate. 

NOTE: MERL data from August 17 to December 6, 1976. Bay data from 
1972-1973 survey. For detailed information on the analysis see Table 24-6. 


significantly different from microcosms 3, 5, 9, and microcosm 3 is 
significantly different from microcosm 9, but the bay stations “Dutch Island,” 
“Southeast Prudence,” “East Prudence” and “Mt. Hope Bridge,” are not 
significantly different from any of the microcosms (Table 24-6). Other mid-bay 
stations are different from some of the microcosms, but no more so than they 
are with regard to each other. 

These intercomparisons between the MERL microcosms and Narragansett 
Bay show that the MERL microcosms diverged surprisingly little from stations 
in the bay or from other microcosms, with regard to concentrations of 
nutrients, chlorophyll, zooplankton biomass and phytoplankton species 
composition during the replicability experiment. 


375 




Table 24-6. A Discriminant Analysis of 9 MERL Microcosms and 13 Bay Stations Using 
Variables Chlorophyll, Zooplankton, Ammonia, Nitrate Plus Nitrite, Phosphate and Silicate from 

August 17 to December 6, 1976 and 1972 Respectively 

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LEGEND: 1 - 9 MERL microcosms, 10 Providence River, 11 Conimicut, 12 Ohio Ledge, 13 Month Greenwich, 14 S. Patience, 15 Quonset, 16 Dutch I 
Mouth W. Passage, 18 Mouth E. Passage, 19 S. Rose Is., 20 S. E. Prudence, 21 E. Prudence, Potters Cove, 22 Mt. Hope Bridge. 





DISCUSSION 


These initial observations provide us with insight into the problems of 
running microcosms in such a way that they are analogous to some natural 
system. The natural system itself is highly variable and difficult to define, 
except within broad limits. Generally, for most of the variables measured, the 
values from the MERL microcosms fell within the ranges observed for adjacent 
Narragansett Bay. We have no evidence that the major features of 
phytoplankton and nutrient dynamics were different from Narragansett Bay. 
This lends support to the hope that the MERL microcosms will be useful 
experimental systems in which investigations will produce results transferable 
to comparable open, natural systems. 

An exception to the generalizations above was the zooplankton abundance. 
The biomass of zooplankton in the MERL microcosms (Figure 24-9) was 
somewhat less than in the bay, especially towards the end of the time period 
considered. This factor is responsible for the microcosms lying somewhat 
outside the fields for the bay data shown in Figures 24-6 and 24-8, because the 
1972-73 bay survey also returned somewhat higher zooplankton biomass 
concentrations than were found in the tanks. We believe that the tendency 
towards low zooplankton biomass was due to an artifact associated with the 
delivery of water to the tanks and that this problem will be rectified by 
subsequent changes to the plumbing. It is therefore premature now to dwell on 
the nature of the differences in zooplankton. 

The general behaviour of the nutrient and the phytoplankton data sets 
(and, to a lesser extent, that of the zooplankton) was most reassuring. No wild 
excursions occurred. The variability in the microcosms was generally similar to 
that in mid-bay stations, and the species abundances were generally similar, 
taking the data as a whole. On the other hand, the quantitative variability of 
the tanks between themselves (as may be inferred by examination of the ranges 
shown in Figure 24-3) violates our usual perception of the way in which 
experimental systems should behave. We expect them to replicate well, so that 
experiments can be performed and the results have good statistical validity. 
The difficulty with replication of nature is that nature herself is highly variable. 
Working with such systems requires that large data sets be obtained, and that 
multivariate statistical techniques be applied to reduce these correlated data 
sets to manageable formats for analysis. 

A possible way of assessing microcosm and natural system behaviour and 
developing a criterion for comparison is to calculate the generalized distance 
between data sets (Blackith and Reyment 1971). The assumptions of 
homogeneity, multivariate normality and linear correlation between variables 
must be met for such a technique to be rigorously applied but, as with all 


377 



Figure 24-9. Zooplankton Biomass in the MERL Tanks and 
in the Bay, During the Fall of 1976. 

NOTE: Solid lines give the range of data from three stations occupied by 
Durbin and Durbin (c.f. Figure 24-4). The solid circles give data from the GSO 
dock. Dashed lines give the range of data from the nine MERL tanks. 


378 




multivariate techniques, some interpretations may be explored even when the 
assumptions are not met. Generalized distances are determined in discriminant 
analysis and the maximum distances for the microcosms and the bay for the 
first axis in Figure 24-8 and Table 24-6 are shown in Table 24-7. These distances 
are much smaller among the replicated microcosms than among bay stations. It 
seems feasible to consider that standard generalized distances exist for natural 
systems, for specific variable sets, which might be compared to generalized 
distances which result during experiments on perturbation and subsequent 
recovery in microcosms. 


Table 24-7. Maximum Generalized Distances and 
Normalized Distances Among the Microcosms and Among 
Bay Stations from August to December 1976 and 1972 
Respectively, from the Discriminant Analysis Shown in 

Figure 24-7 and Table 24-6. 



max 

Normalized 
D-j max 

Microcosms: 

2 vs 3 

0.9 

3 

2 vs 5 

0.7 

2 

2 vs 9 

0.3 

1 

3 vs 9 

0.6 

2 

Bay Stations: 

Prov. River vs mouth E. Passage 

4.4 

14 

Ohio Ledge vs mouth E. Passage 

3.0 

9 

Ohio Ledge vs mouth W. Passage 

1.9 

6 


CONCLUSIONS 

The nine MERL microcosms operated during the 4-month replicability 
study were generally as similar to each other as they were to adjacent areas of 
Narragansett Bay using nutrient and phytoplankton data sets for comparison. 

Zooplankton abundance in the MERL microcosms was somewhat low but 
this was probably caused by an artifact that can be removed. 

Multivariate statistical techniques seem essential to the comparison of the 
large and heterogeneous data sets generated in such studies as this. 


379 






The similarity in the behaviour of the microcosms and Narragansett Bay 
gives some confidence that these systems will be good experimental tools lor 
ecological and biogeochemical experiements. 

ACKNOWLEDGMENTS 

This work was supported by grant No. R803902020 from the 
Environmental Protection Agency. We thank Ann and Ted Durbin for allowing 
us to use their phytoplankton counts and zooplankton biomass data in Figures 
24-4 and 24-9, and Scott Nixon for helpful discussion. 

REFERENCES 

1. Blackith, R. E. and R. A. Reyment. 1971. Multivariate Morphometries. 
Academic Press, London. 412 pp. 

2. David, M., C. Campiglio and R. Darling. 1974. Progress in R- and Q-mode 
Analysis: Correspondence Analysis and its Application to the Study of 
Geological Processes. Can. J. Earth Sci. 11:131-146. 

3. Davies, J. M., J. C. Gamble and J. H. Steele. 1975. Preliminary Studies with 
a Large Plastic Enclosure. In: L. E. Cronin, (ed.) Estuarine Research, Vol. I, 
pp. 251-264. 

4. Furnas, M. J., G. L. Hitchcock and T. J. Smayda. 1976. 
Nutrient-phytoplankton Relationships in Narragansett Bay during the 1974 
Summer Bloom In: Estuarine Processes Vol. I, Uses, Stresses and 
Adaptations to the Estuary, Martin Wiley (ed.) Academic Press, N. Y. pp. 
118-134. 

5. Holm-Hansen, 0., C. T. Lorenzen, R. W. Holmes and J. D. H. Strickland. 
1965. Fluorometric Determination of Chlorophyll. J. Cons. Perm. Int. 
Explor. Mer 30:3-15. 

6. Rremer, J. N. 1975. Analysis of a Plankton-Based Temperate Ecosystem: 
an Ecological Simulation Model of Narragansett Bay. Ph.D. Dissertation, 
Univ. of Rhode Island. 

7. Kremer, J. N. and S. W. Nixon. 1977. A Coastal Marine Ecosystem, 
Simulation and Analysis. Springer-Verlag, New York, 200 pp. 

8. Lacaze, J. C. 1975. Experiences de Pollution en Ecosystemes Marine 
Contrdles. Applications aux Produits Petroliers Oceanis 2, (Supp. 1): 
1-115. 


380 


9. Menzel, D. W. 1977. Summary of Experimental Results: Controlled 
Ecosystem Pollution Experiment. Bull. Mar. Sci. 27:142-145. 

10. Menzel, D. W. and J. Case. 1977. Concept and design: Controlled 
Ecosystem Pollution Experiment. Bull. Mar. Sci. 27:1-7. 

11. Nie, N. H., C. H. Hull, J. G. Jenkins, K. Steinbrenner, and 0. H. Bent. 
1975. Statistical Package for the Social Sciences. 2nd edition. McGraw Hill 
Book Co., New York, pp. 434-462. 

12. Nixon, S. W., C. A. Oviatt and S. S. Hale. 1976. Nitrogen Regeneration and 
the Metabolism of Coastal Marine Bottom Communities. In: The Role of 
Terrestrial and Aquatic Organisms in Decomposition Processes, The 17th 
Symposium of the British Ecological Society. J. M. Anderson and A. 
MacFadyen (eds.). Blackwell Scientific Publications. London pp. 269-283. 

13. Odum, H. T., W. L. Siler, R. J. Beyers, and N. Armstrong. 1963. 
Experiments with Engineering of Marine Ecosystems. Contrib. in Mar. Sci. 
9:373-403. 

14. Perez, K. T., G. M. Morrison, N. F. Lackie, C. A. Oviatt, S. W. Nixon, B. 
Buckley, and J. F. Heltshe. 1977. The Importance of Physical and Biotic 
Scaling to the Experimental Simulation of a Coastal Marine Ecosystem. 
Helgolander Wiss. Meeresunter, 30(1-4): 144-162. 

15. Pielou, E. C. 1969. An Introduction to Mathematical Ecology, 
Wiley-Interscience, New York, 286 pp. 

16. Pilson, M. E. Q., G. A. Vargo, P. Gearing and J. N. Gearing. 1977. The 
Marine Ecosystems Research Laboratory: A Facility for the Investigation 
of Effects and Fates of Pollutants. Proc. 2nd National Conf. Interagency 
Energy/Environment R & D Program, Wash., D.C. 

17. Smetacek, V., B. Von Bodungen, K. Von Brockel and B. Zeitzschel. 1976. 
The plankton tower. II. Release of Nutrients from Sediments due to 
Changes in the Density of Bottom Water. Mar. Biol. 34:373-378. 

18. Strickland, J. D. H. and T. R. Parsons. 1972. A Practical Handbook of 
Seawater Analysis. Fish. Res. Board ol Can. Bull. 167, 2nd Edition. 310 

pp. 

19. U. S. Army Corps of Engineers. 1959. Contamination Dispersion in 
Estuaries. Narragansett Bay, Hydraulic Model Investigation. U. S. Army 
Eng. Waterways Exp. Sta. Misc. Paper 2-332, Report 2, Vicksburg, MI. 


381 


TURBULENT MIXING IN MARINE MICROCOSMS— 
SOME RELATIVE MEASURES AND ECOLOGICAL 

CONSEQUENCES 


Scott W. Nixon, Candace A. Oviatt and Betty A. Buckley 
Graduate School of Oceanography 
University of Rhode Island 
Kingston, R.l. 02881 


ABSTRACT 

The effect of turbulent water motion on pelagic organisms has seldom been 
studied. Nevertheless, a consideration of the theory of turbulent energy flux as 
well as the few bits of empirical data which do exist suggest that it may be a 
factor of some importance for marine plankton, and that turbulence may 
influence the growth, metabolism, and behavior of pelagic species as well as 
their spatial distribution. This paper reports the results of a series of turbulence 
experiments carried out over an annual cycle using small (150 1) laboratory 
microcosms designed as analogues of Narragansett Bay, R.L (U.S.A.). 
Turbulence levels in the mocrocosms and in the natural system were 
characterized using conventional (neighbor diffusivity, vertical eddy diffusivity, 
energy flux) parameters as well as a number of relative measures of water 
mixing (dye dissipation, CaSO^ dissolution rate, gas exchange coefficients). 
The response of phytoplankton and zooplankton populations to varying 
turbulence levels was dramatic during warmer months, but absent or unclear in 
winter. The results suggest that while phytoplankton may be stimulated by 
higher turbulence levels, at least in warmer water, the response of the 
zooplankton is quite the opposite during these periods. It is not clear if the 
response of the phytoplankton reflects a decline in grazing pressure or a real 
enhancement of growth. The problem is complex and deserves considerable 
further study both in the field and in the laboratory. 

INTRODUCTION 


“.. . diffusion is confusion. Only Maxwell’s Demon 
really knows what’s going on.” 

Akira Okubo (1971), 

Horizontal and Vertical Mixing in the Sea 


382 


A Problem of Size and Scale 


The increasing use of relatively small experimental ecosystems or 
microcosms in marine research has raised a number of interesting questions 
related to the importance of size or scale in natural, as well as experimental 
systems. In pelagic marine environments, one of these questions for which we 
have very little relevant information is the importance of turbulence in the 
water. The relationship between turbulence and scale was first formalized by 
Richardson (1926), who distinguished between classical Fickian diffusion (in 
which scale is not a factor) and the mixing that is characteristic of turbulent 
fluids such as the sea. While the nature of turbulence is extremely complex, 
Richardson’s concept of turbulent energy passing through a series of 
progressively smaller eddies from the wavelength at which it is put into the 
fluid until it is ultimately dissipated in viscosity has continued to prove 
valuable in studying the mixing of marine waters (Okubo 1971). In natural 
systems, turbulent energy is added at a rather large scale by winds, tides, and 
major currents. Since none of these is usually effective in microcosm tanks or 
bags, some artificial means of introducing turbulent energy at smaller scale may 
be required to develop pelagic ecosystems that are credible experimental 
analogs of the “real world” (Perez et al 1977). 


There are at least two aspects to the turbulence problem, one involving the 
actual distribution of organisms, particles, or dissolved constituents in the 
water — the problem of patchiness (see Steele 1974), and the other involving 
the metabolic or behavioral responses of organisms to water turbulence. The 
study of plankton patches usually concerns water masses on a scale larger than 
the largest microcosms yet developed (1300 m^, 10 m dia.; see Menzel and 
Case 1977), and it is generally conceded that this aspect of the ecology of 
marine waters is not well represented in microcosm experiments. The 
importance of this omission is not yet known. While there have been numerous 
studies which have documented the response of sessile plants and animals to 
the special case of turbulence in flowing water (Fox et al 1935; Kerswill 1949; 
Whitford 1960; Jaag and Ambiihl 1963; Whitford and Suchumacker 1964; 
Mclntire 1966; Westland 1967; Nixon and Oviatt 1971), the responses of 
pelagic organisms to small scale turbulent energy have received much less 

attention. 


With the exception of an older qualitative study of the morphological 
response of Daphnia to water motion by Brooks (1947), the recent work by 
Pasciak and Gavis (1975) on the relationship between turbulence and nutrient 
uptake by phytoplankton, and our own studies on marine plankton in 
laboratory microcosms (Perez et al 1977), the effect of water turbulence on 
the growth and metabolism of planktonic organisms is almost totally unknown. 
While this situation is largely a result of the difficulties involved in measuring 


383 



turbulent energy levels in the laboratory or in the field, it may also reflect the 
feeling of many ecologists that the small size of plankton generally places them 
below the size scale at which turbulence is “felt”. Both of these considerations, 
along with technical difficulties, have caused almost all marine microcosm 
studies to neglect turbulence as a factor in their experimental design. In the 
simplest terms, the justification appears to have been that since turbulence is 
difficult to measure, hard to mimic, and of unknown importance, it was 
reasonable to avoid the problem of deciding on how to include it in 
microcosms. There is a certain amount of appeal to this argument, especially 
since there are so many other problems to be resolved in developing a 
microcosm. However, the evidence in the papers cited above, as well as the 
experience of anyone who has tried to culture or maintain phytoplankton and 
zooplankton in the laboratory, suggest that turbulence is an important 
consideration in pelagic systems. Our earlier experiments with turbulence in 
marine microcosms also indicated that the scaling of mixing energy in 
laboratory tanks can dramatically influence the results of phytoplankton and 
zooplankton growth studies in the microcosms (Perez et al 1977). The 
argument about plankton being too small to “feel” turbulence is also 
questionable. 

Turbulence 


Following Richardson (1926), Richardson and Stommel (1948), Stommel 
(1949), Batchelor (1950) and others, the flow of turbulent energy from large 
scale motion is passed down through successively smaller eddies until it is 
dissipated in viscosity. Above a certain size, the energy content of eddies is 
solely a function of their size (k) and the rate of energy flux (e) through the 
system. Below this critical size, defined by 


k = 


*3 1/4 


6 



where v = the kinematic viscosity 

e = the energy flux per unit mass 
k = upper limit of the kolmogoroff viscous zone 

viscous forces become important and the energy content decays more rapidly 
with decreasing size as energy is dissipated. In order to give some feeling for the 
scale involved, it is possible to estimate k for the West Passage of Narragansett 
Bay using a value, for the energy dissipation of 4.3 x 10 13 ergs sec' 1 (Levine 
and Kenyon 1975) and an approximate volume of 7.2 x 10 8 m 3 . The result 
su 88 ts ts that k is on the order of 0.06 cm. While this is larger than individual 
phytoplankton cells found in these waters (<0.01 cm), it is about the size of 


384 



many of the diatom chains found in the bay (~0.05-0.1 cm) and smaller than 
the dominant zooplankton. Moreover, even below this size, the effect of 
turbulent energy persists. As the time averaged flux of energy through larger 
eddies is increased, the viscous shear in the Kolmogoroff zone will also increase 
and this increase in shear will be felt even at the very small scales seen by the 
plankton. Moreover, as the energy flux through larger eddies increases, the 
upper size limit of the Kolmogoroff zone will decrease, so that larger plankton 
will begin to experience direct turbulent effects. 


In addition to simple mechanical effects, such as the disruption of feeding 
or copulation by zooplankton, this turbulent energy flux at small scale may 
influence the plankton (or other particles) in at least two ways. Around any 
given cell of size fi, there will exist a thin laminar boundary layer in which 
Fickian or molecular diffusion must be relied upon to transport dissolved gases, 
nutrients, waste products, etc. Since molecular diffusion is much slower than 
turbulent diffusion, this is often the rate limiting step in exchange processes 
between the cell and the surrounding medium. As the turbulent energy flux in 
the medium increases, however, the water just outside of the boundary layer is 
renewed more rapidly, with the renewal rate being proportional to: 


el/2 

4v 



This increase in renewal rate tends to maximize the concentration gradient 
across the laminar boundary layers and, thus, the diffusion of materials across 
the layer. In addition, the increase in renewal rate by turbulent velocity will 
also decrease the thickness of the boundary layer itself, since the boundary 
layer thickness is proportional to: 


1 




Again, the reduced thickness of the laminar layer will increase the exchange 
rate of materials between the particle and the medium. 

While the cascade of turbulent energy through successively smaller eddies 
has been studied frequently in the sea (Okubo 1971), the emphasis in the field 
has generally centered on measurements of eddies larger than 10 m. The nature 
of the turbulent energy spectrum in small experimental ecosystems has only 
recently begun to receive attention (Boyce 1974, Steele et al 1977, Gust 


385 




1977). While the results of Gust’s study are restricted to the specific flexible 
chambers and conditions of his measurements, they show quite convincingly 
that it is possible to obtain a small scale turbulent spectrum in a chamber that 
is similar to that found in the surrounding coastal waters. Unfortunately, 
almost all of these turbulence measurements were made in a metabolic 
chamber used with benthic algae rather than in plankton studies, and no 
biological data were included. 

This paper reports the results of a number of turbulence experiments carried 
out at different times of the year using coupled benthic-pelagic microcosms 
designed as analoges of Narragansett Bay, R.I. While some of the data from 
experiments conducted during the spring have been reported previously (Perez 
et al 1977), we have now carried out identical studies during winter and 
summer months. In addition, we have explored in this paper a number of 
techniques for characterizing the turbulent mixing levels in the microcosms and 
compared them to the dissolution rate measurements used previously (Oviatt et 
al 1977, Perez et al 1977). Finally, we have attempted to carry out 
experiments to test our earlier conclusion that phytoplankton and zooplankton 
respond independently to different turbulence levels. The impression that the 
phytoplankton and zooplankton were not coupled in their response to 
turbulence was based on indirect evidence (Perez et al 1977) and we felt it 
desirable to test this conclusion directly by adjusting the levels of zooplankton 
in replicate tanks and observing whether concommitant but opposite changes 
would occur in the phytoplankton. 


METHODS 
The Microcosms 

The microcosms used in these experiments have been described in detail in 
earlier papers (Perez et al 1977; Oviatt et al 1977 and in press). Each 
microcosm consisted of a 166 liter plastic tank containing 150 1 of water (0.7 
m deep) collected by bucket from the lower West Passage of Narragansett Bay. 
This area of the bay shows a well mixed water column about 8 m deep with 
salinity between 28-3 l 0/ oo throughout the year. Characteristics of the bay have 
been described in some detail by Kremer and Nixon (1978). The microcosms 
were maintained in a running sea water bath in the laboratory near field 
temperatures and illuminated for the appropriate natural photoperiod by 
Westinghouse Cool White fluorescent lights. The response of the microcosms to 
light input is complex and the choice of a value for any particular experiment 
is difficult (Nixon et al, in press). The experiments described here were carried 
out at 5-25 ly/day, values considerably below the average tight energy found in 
the water column of the bay. 


386 


Each microcosm also contained an opaque plastic box (167 cirr) of intact 
bay sediment and associated benthos. This size produced the same sediment 
surface to water volume ratio as found in the bay. Water from the pelagic phase 
of the microcosm was moved through the box and over the sediment by 
vacuum pump so as not to damage the plankton. The inside walls of the tanks 
were cleaned regularly to prevent fouling, and organic matter settling on the 
bottoms of the tanks was collected and placed in the sediment 6oxes. In all of 
the turbulence experiments, it is important to note that the artificial nature of 
the “bottom” community isolated it from the turbulent energy of the water, 
except as the benthos might respond to changes in the plankton. However, 
even in tanks that were unstirred, the benthic box pumps provided some very 
gentle circulation for the pelagic community, since the flow rate used was 
capable of putting 150 liters of water through the box about three times each 
day. Additional mixing was contributed by the approximately daily wall 
cleaning and by the addition of 10 liters of bay water to each tank three times 
each week. The latter was maintained so that the microcosms functioned as 
open systems with a flushing rate similar to that of Narragansett Bay. 

Different turbulence levels were imposed on the microcosms by leaving 
them unstirred except for the benthic pump and cleaning operations or by 
mixing them with plastic mesh paddles of 0.14 or 0.07 m area. The 
opening size of the plastic grid in the paddles was 1.2 cm x 1.2 cm. The paddles 
were driven at 32 rpm by an electric motor connected to all the paddle shafts 
by a chain, thus producing identical rotation rates in all of the tanks. Each 
paddle was rotated in one direction for 30 sec., then stopped for 6 sec., then 
reversed for 30 sec. in a continuous cycle. 


Turbulence Measurements 

Vertical Eddy Diffusivity 

We attempted to obtain a variety of both relative and absolute 
measurements of turbulent mixing in the microcosms and in Narragansett Bay. 
In some cases, such as the estimation ol a vertical eddy diffusivity, the 
techniques used were conventional. A small amount (~2 ml) of Rhodamine-WT 
dye was dissolved in sea water (1:100) and released “instantaneously” at 
mid-depth in the microcosms. Near-surface and near-bottom water samples 
were then collected at short intervals (2-5 min.) and the concentration of dye 
determined fluorometrically. The rate-of-change in concentration in both sets 
of samples was virtually identical, indicating that the tanks were mixed 
uniformly up and down, and that the vertical eddy diffusivity could be 

estimated by 


387 


(4) 


Z 2 


2t 

2 -1 

D y = vertical eddy diffusivity, cm sec 
Z = distance between relative point and measurement 
point (1/2 depth in this case), cm 
t = time for the concentration to asymptote, sec 

The average value of this parameter for Narragansett Bay has been computed 
by Hess (1976) using a detailed numerical hydrodynamic model. 

The Horizontal Turbulent Field 

Determination of the horizontal turbulent component was more difficult. 
At one extreme, we measured the time it took for small dye patches (0.5 ml of 
1:100 dye in seawater) to disperse in the microcosms and in the bay under a 
range of conditions. This approach was simple, rapid, and with enough 
replication and a constant observer, it gave a good (low variance) relative 
measure of horizontal mixing rates. Unfortunately, it is also a bit subjective 
and qualitative and cannot be expressed directly as a standard hydrodynamic 
parameter. 

In an attempt to overcome these limitations, we have also obtained 
measurements of neighbor diffusivity (Richardson, 1926; Stommel, 1948) and 
the flux of turbulent energy along two arbitrary perpendicular coordinates 
(Batchelor, 1950) using the relative motions of pairs of small floats with a 
range of distances separating them. The measurements were made by releasing 
several dozen floats and then photographing them from a fixed position at 
short time intervals. The size of the floats used and the length of the time 
interval were varied somewhat according to the scale of the turbulent eddies of 
interest. In the West Passage of Narragansett Bay, larger scale mixing (1-25 m) 
was studied using colored balloons filled with fresh water so that they floated 
just beneath the surface. These floats were dispersed from a small boat and 
photographed every few minutes from a high bridge. Smaller scale eddies 
(1-200 cm) were studied in the field using small (~0.5 cm) colored plastic 
beads that were released from the end of a pole off the stern of a small boat 
and photographed every few seconds using a 16 mm movie camera operated 
from the flying bridge of the boat. The beads and the movie camera were also 
used in the microcosms. In all cases, floating rods of standard length were 
included in each photograph to give an accurate scale. 

After they were developed, the films of the floats were put through a 
microfiche reader for enlargement. Large numbers of pairs of floats were 


388 



selected at various distances of separation to be followed from frame to frame 
over time. In each frame the scalar distance between the floats as well as their 
separation along two perpendicular vectors (x and y) was obtained. These data 
were then analyzed using the relationship given by Batchelor (1950). 



where 


is the initial scalar distance between floats 

£ is the initial distance between the floats 
A 1 

projected on the x axis 

C is the distance between the floats after some 

"z • 

time, t, projected on the x axis 
e is the rate of turbulent energy flux 


The same operation was carried out for the *y* vector which should yield a 
similar value if the turbulent field is isotropic. Unfortunately, it is extremely 
difficult to extend the analysis to a third dimension and it is not practical to 
use the method to explore the horizontal turbulent field below the surface. 


Additional Relative Turbulence Measures 


As discussed earlier, the flux of turbulent energy influences the rate at 
which materials may be exchanged across a laminar boundary layer. This 
suggests that the dissolution rate of a solid substance placed in the water may, 
at least to some degree, be a function of the turbulent energy of the fluid. The 
importance of the turbulent effect should be greatest for materials that are 
near saturation in sea water. After some exploration, we have found that the 
mineral gypsum (CaS0 4 ) is particularly well suited for this purpose. It is easily 
obtained, inexpensive, and a large number of uniform pieces can be cut from a 
single rough block. The dissolution rate is influenced somewhat by temperature 
and salinity, but these relationships are easily established in the laboratory in 
order to compare measurements made under different conditions. Since the 
rate of weight loss is also a function of size, we have found it best to use 
standard pieces of gypsum measuring ~2.5 x 1.8 x 0.7 cm with an initial 
weight of about 6-8 gms. Blocks of this size are suitable for making 
measurements of weight loss over periods ranging from about 6-24 hrs. 
Replication appears to be quite good and duplicate blocks are hung off a fixed 
or free floating line to obtain a vertical profile of dissolution rate. 
Unfortunately, however, the CaS0 4 dissolution rate may provide only a 
relative measure of turbulent energy and it is not clear if it is possible to relate 


389 






it to any more conventional physical measurements. It is also not clear how 
one might separate vertical and horizontal components of mixing using the 
blocks. 

An additional indirect estimate of mixing in the microcosms and in the field 
was obtained by measuring the diffusion coefficient for oxygen across the 
air-water interface. The measurements were made using a small floating plastic 
dome from which virtually all of the oxygen was displaced by nitrogen gas. The 
partial pressure of oxygen in the dome and in the water was then monitored 
over time and the flux of oxygen from the water into the dome calculated. 
Since the flux is a product of the gradient in partial pressure and the diffusion 
coefficient, it was possible to obtain the coefficient from such a data set. 
Measurements with domes containing turbulent or still air have confirmed that 
the diffusion coefficient is largely a function of turbulence in the liquid phase, 
and that the effect of wind is felt through its influence on water mixing. 


RESULTS 

Turbulence Levels Obtained 

The results of the various turbulence measurements lead us to be 
particularly sympathetic to Okubo’s lament that “diffusion is confusion.” In 
spite of, or perhaps because of the fact that a large number of floating pair 
observations were made in Narragansett Bay and in the microcosms, there was 
a very large amount of scatter in these data. As a result, the calculation of 
neighbor diffusivity (F) and energy flux (e) was subject to a large uncertainty 
and there is some question about how meaningful the numbers may be. While 
the values of F tended to decrease approximately according to the 4/3 law 
(Richardson 1926; Stommel 1948), it also appeared that e had a tendency to 
fall off with size. The latter result is disturbing since the theoretical framework 
for the computation suggests that e should be constant at steadystate in the 
inertial range between the size at which energy is put into the system and the 
viscous zone in which it is dissipated. 


Functional regressions (Ricker 1973) relating F to scale are given below. In 
general, both the neighbor diffusivity and the energy flux indicated that the 
turbulence levels in the microcosms with one paddle were appreciably higher 
than found in the bay or in the mocrocosms with one half paddle and no 
paddle (Table 25-1). The F Values did not show a great difference between the 
half paddle microcosms and the bay and the very large variance associated with 
the calculation of e in the half paddle tanks made it difficult to see any clear 
differences in terms of energy flux. 


390 


Table 25-1. Estimates of Turbulent Energy 
Dissipation Rates in the Experimental Microcosms 
and in Some Natural Marine Waters 


Microcosms with one paddle 

Microcosms with half paddle 

Microcosms with no paddle 

Narragansett Bay, West Passage 
measured at different times 

Narragansett Bay, West Passage 
estimated from tidal currents, 

( Levine and Kenyon, 1975) 

Irish Sea, estimated from tidal 
currents (Taylor, 1919) 

N.W. Pacific coastal water, U.S.A. 
surface 30m, calcualted from 
changes in microstructure 
(P.W. Nasmyth in Gregg 1973) 


e, cm' 



1.44 

0.03 

*0 


0.05, 0.07, 0.11, 
0.17, 1.0 


0.21 


0.08 


0.02 


Open sea, surface mixed 
layer (50 m) (Gregg 1973) 


0.002 


2 -1 

Functional Regressions for F, cm~ sec 

West Passage F = 0.007 Cl.89 (6) 

Microcosms with one paddle F = 0.108 Cl.38 
with half paddle F = 0.013 Cl.73 

The simpler measurements of horizontal dye patch dispersion suggested that 
while the one paddle microcosms were considerably more turbulent than those 
with half a paddle, both were more rapidly mixed than the bay (Table 25-2). 
This same trend with respect to differences between the whole paddle and half 
paddle microcosms was also shown by the weight loss ot gypsum blocks and 
the gas exchange measurements (Table 25-2). However, both of these 
parameters indicated substantially higher mixing rates in the bay than in any ot 


391 









Table 25-Z Relative Turbulence Measurements 
in the Experimental Microcosms and in Narragansett Bay 


Vertical Eddy Diffusivity 


2 -1 
K, cm sec 

Microcosms with one paddle 3.6 - 3.8 

Microcosms with half paddle 2.2 - 3.8 

Microcosms with no paddle * 0 

Narragansett Bay, mean for the 

West Passage (Hess 1976) 5 


Rates of Horizontal Dye Patch Dispersion 


Time to Disperse, sec 

Microcosms with one paddle (N=7) 5.4 ± 1.2 

Microcosms with half paddle (N=7) 8.4 ± 1.2 

Microcosms with no paddle (N=1) » 900 

Narragansett Bay, West Passage 

day 1 (N=10) 18.0 ± 5.4 

day 2 (N=6) 13.4 ± 1.9 

day 3 (N=6) 17.5 ± 2.5 


CaSO^ Dissolution Rate 


Microcosms with one paddle 
Microcosms with half paddle 
Microcosms with no paddle 


Weight Loss, % hr' 1 

1.83 ± 0.20 
0.73 ± 0.36 
0.27 ± 0.05 


Narragansett Bay, West Passage, 
mean for vertical profiles 


3.30 ± 0.60 
4.39 ± 1.10 


C >2 Diffusion Coefficient at the Air-Water Interface 

K, juM m~2 hr~^ atm'^ 

Microcosms with one paddle 27 

Microcosms with half paddle 8 

Microcosms with no paddle 2 


Narragansett Bay, West Passage 

calm day 30 

windy day 137 

windy day 125 


392 








the microcosms. To some extent this may reflect the fact that the floating pair 
measurements and dye patches respond to the horizontal turbulent field and 
the gypsum and gas exchange are also influenced by vertical motion. The 
rotating plastic paddles appeared to add a lot of horizontal mixing energy to 
the tanks, but the vertical eddy diffusivity in the microcosms was lower than 
Hess (1976) calculated for Narragansett Bay (Table 25-1). 

While none of these measurements allows us to make a very convincing 
absolute comparison of turbulent energy in the microcosms with that of the 
bay, it does seem clear that the full paddle, half paddle, no paddle 
configuration provided quite different turbulent water regimes in the 
microcosms. Since the input of turbulent energy to Narragansett Bay must vary 
considerably during the tidal cycle and from day-to-day according to the 
winds, it seems reasonable that the natural pelagic community may well 
experience all of the turbulent conditions used in the microcosms. For 
comparative purposes, it is interesting to note that all of the methods used for 
measuring turbulence except the determination of neighbor diffusivity and 
energy flux (e) indicated that even the full paddle configuration was low 
relative to the bay. 

Response of the Plankton 

The first turbulence experiment was carried out during the month of April 
when water temperatures in the microcosms ranged from 8 to 12°C. The 
standing crop of phytoplankton as indicated by chi a increased dramatically in 
the one paddle and half paddle treatments compared with the unstirred tanks 
(Figure 25-1). A number of cursory analyses of water samples did not indicate 
that there were any major shifts in species composition in the different tanks. 
However, there were also marked and significant differences (Perez et al 1977) 
among treatments in the numbers of Acartia clausi, the dominant zooplankton 
in the microcosms and in the bay (Figure 25-2). While the rapid increase in 
phytoplankton in the one paddle tanks began almost immediately, Acartia 
nauplii did not really start to decline until after 10 days. In fact, a portion of 
the decline in nauplii between 10 and 16 days was simply due to growth of the 
animals into juveniles (Figure 25-2). An analysis of covariance was performed 
to establish whether the changes in phytoplankton density could be attributed 
to changes in zooplankton density the covariate, total grazers was found to be 
non-significant. This meant that the inverse relationship expressed by 
zooplankton and phytoplankton to water turbulence was due to a direct 
pattern than the indirect effect of water turbulence. In fact, an analysis of 
covariance on the mean algal standing crop during the experiment indicated 
that interactions with the total numbers of grazers in the microcosms (the 
covariate) was not significant (Perez et al 1977). It is possible, however, that 
the zooplankton present did not feed as effectively in the more turbulent 


393 


20 


t 


15 


!_I 

O' 



0 



DAYS AFTER 5 APRIL 


Figure 25-1. Chlorophyll Concentrations (In Vivo Fluorescence) 
in the Microcosms and in the Lower West Passage of Narragansett 
Bay During the First Turbulence Experiment Begun April 5, 1976. 


NOTE: Data points are the mean of duplicate tanks. 


tanks. It is striking that the zooplankton standing crop with one paddle was 

» 

very similar to that found during the same period in the bay, while the 
phytoplankton populations in those tanks reached levels 3 times greater than 
found in the bay with similar zooplankton numbers. Conversely, the low and 
relatively constant phytoplankton standing crops in the unstirred microcosms 
were almost identical to that found in the bay, but much larger zooplankton 
populations were sustained, at least for 15 days, in the microcosms. It may be 
that the plankton in the microcosms escaped a significant grazing and/or 
predation pressure that was important in setting the standing crop maintained 
in the field. This experiment was repeated during May with virtually the same 
results. 


The next turbulence experiment was not begun until December, when water 
temperatures ranged from 1 to 6°C and the standing crops of phytoplankton 
were low. The experiment was designed to explore not only the effect of 
turbulence, but also the interactions of turbulence with light and nutrient 
enrichment. Again, the standing crop of phytoplankton was significantly (0.05 
level) higher in tanks mixed with a full paddle, though the effect was not as 
dramatic as in the earlier runs (Figure 25-3). The response to light was not 
significant at the 0.05 level but was significant at the 0.10 level. The response 
to turbulence was highly significant (greater than the 0.01 level) (Figure 254). 


394 













Figure 25-2. Numbers of Adult, Juvenile, and Nauplii 
Acartia clausi in the Microcosms and in the Lower West Passage 
of Narragansett Bay during the First Turbulence Experiment. 

NOTE: Data points are the mean of duplicate tanks. 


395 





















PHYTOPLANKTON, cells ml' 





Figure 25-3. Phytoplankton Cell Counts in the Microcosms and 
in the Lower West Passage of Narragansett Bay During the 
Turbulence-Light Interaction Experiment Begun December 9, 1976. 

NOTE: Data points are the mean of duplicate tanks. 


396 























PHYTOPLANKTON, cells ml' 1 x 10 



MIXING, No. of Paddles 


Figure 25-4. Average Phytoplankton Population Levels During 
the 34 Day Turbulence-Light Experiment. 

NOTE: Data points are the mean of duplicate tanks. 


397 












The interaction of light and turbulence was not significant nor was the effect 
of ammonia enrichment. However the ammonia addition brought the 
concentration in the tanks from ~3/iM to so that the plankton were 

never seriously nutrient limited. It was also interesting that there was no 
response of the phytoplankton in the unmixed microcosms to increased light, 
while there was a clear increase in the stirred tank populations with higher light 
levels (Figure 25-4). The numbers of zooplankton, again dominated by A. 
clausi, were very low throughout the experiment (Nauplii ^10/L; juveniles 
«5/L) and no dramatic differences among treatments developed. However, the 
mean numbers of nauplii and juveniles observed during the experiment were 
higher in the tanks with no paddle and lowest in the tanks with one paddle. 
Analysis of the data showed that this difference in the means was statistically 
significant (a:0.05) and that there was no significant interaction of nauplii, 
juveniles, or adults with light intensity. There was no statistically significant 
difference in the mean number of adults in the different turbulence levels. 

In order to find out if turbulence had a direct stimulating effect on 
phytoplankton, two experiments were carried out during January and 
February in which an attempt was made to remove zooplankton from some of 
the microcosms by filtering the water through a #20 (80 ju) net. This was 
effective in reducing the zooplankton levels by about 70 percent in the first 
experiment and by about 90 percent in the second. In addition, light levels 
were increased from 6 ly/day during the January run to 16 ly/day in February 
and ammonia was added to all tanks at the start of the second experiment in an 
attempt to stimulate vigorous phytoplankton growth. Temperatures ranged 
from 0-0.5°C during the first experiment and from 0-3°C during the second. 

The results of the first experiment showed no significant effect of 
turbulence on the numbers of phytoplankton or zooplankton in the 
microcosms (Figures 25-5 and 25-6). The lack of turbulence effect on the 
phytoplankton was observed in tanks with and essentially without zooplankton 
(Figure 25-5). It is interesting to note that the variation in zooplankton 
numbers by a factor of about 3.5 had no significant effect on the levels of 
phytoplankton, probably due to low temperatures and therefore reduced 
grazing rates. 

When the experiment was repeated a month later with higher light and 
nutrients, a phytoplankton bloom was produced during the first week in all of 
the microcosms (Figure 25-7). During this period there did not appear to be 
any effect of the turbulence on phytoplankton growth either with or without 
zooplankton. Moreover, the grazing pressure of the small number of A. clausi 
in the unfiltered water (~15 animals/L) at these low temperatures had little or 
no effect on the bloom. However, the bloom declined much more slowly in the 
unstirred microcosms, so that after 10-15 days the standing crops in the 


398 


PHYTOPLANKTON, cells 



WITHOUT ZOOPLANKTON 



o 5 10 15 20 

DAYS AFTER 19 JANUARY 

Figure 25-5. Phytoplankton Cell Counts in the Microcosms and 
in the Lower West Passage of Narragansett Bay During the 
January 1977 Turbulence Experiment. 


NOTE: Data points are the mean of duplicate tanks. 


399 









c/a usi 


20 




Figure 25-6. Numbers of Acartia clausi nauplii and Juveniles 
in the Unfiltered Microcosms and in the Lower West Passage of 
Narragansett Bay During the January Turbulence Experiment. 

NOTE: Data points are the mean of duplicate tanks. 


400 








30 

25 

20 

15 

io' 

5 

. 0 

25 

20 

15 

10 

5 

0 


BAY- 

NO PADDLE o- 


I PADDLE *■ 


WITH 

ZOOPLANKTON 



WITHOUT 

ZOOPLANKTON 





l 


1 


JL 


5 10 15 20 25 

DAYS AFTER 8 FEBRUARY 


30 


7. Phytoplankton Cell Counts in the Microcosms and 
ower West Passage of Narragansett Bay During the 
February 1977 Turbulence Experiment. 

OTE: Data points are the mean of duplicate tanks. 


401 















turbulent tanks were markedly lower both with and without zooplankton 
(Figure 25-7). Statistical analysis of the mean numbers of cells in each 
treatment during the experiment showed that the average standing crop of 
phytoplankton was significantly higher (a = 0.05) in the unstirred tanks. Again, 
this is clearly the reverse of the pattern found in the first three experiments, 
but repeats the trend suggested by the January run (Figure 25-5). There were 
no significant differences in the new numbers of zooplankton between the two 
turbulence levels during this experiment, with both showing small populations 
that fluctuated between about 5-15 animals per liter. This is the first 
experiment in which the phytoplankton showed a significant response to 
turbulence (albeit opposite to that found previously) but zooplankton numbers 
did not. Again, it is interesting to note that an almost 9 fold increase in 
zooplankton numbers did not result in any statistically significant decline in 
the numbers of phytoplankton. 

We attempted to repeat the zooplankton removal experiment during July of 
the following summer with water temperatures between 19-20.5°C. However, 
the hatching and development rate of zooplankton eggs and nauplii is so rapid 
at the higher temperatures that it was virtually impossible to reduce the 
numbers of zooplankton very much by the filtration method used. 
Nevertheless, the results were interesting. The pattern found in the first three 
experiments emerged once again, with phytoplankton growth clearly enhanced 
by the turbulent mixing and zooplankton surpressed (Figures 25-8 and 25-9). 

A final experiment was carried out during August in which the interaction 
of turbulence and water turnover rate in the microcosms was explored. Water 
temperatures varied between 19-21°C. While there was no significant effect of 
turnover rate on the plankton, the same statistically significant stimulation of 
phytoplankton growth was found in the stirred microcosms where zooplankton 
significantly declined by a factor of 2-3 (Figure 25-10). The ten-fold increase in 
phytoplankton associated with somewhat more than a halving of the 
zooplankton in the turbulent microcosms may reflect the tight coupling of 
these two compartments that has been suggested in numerical simulations of 
the summer plankton (Rremer and Nixon 1978). This result contrasts with our 
earlier experiments carried out at lower temperatures in which significant 
reductions in zooplankton numbers had no significant effect on the mean 
phytoplankton standing crop. 

DISCUSSION 

The Importance of Turbulence 

It seems clear that the presence or absence of turbulent mixing in the 
microcosms had a significant influence on the abundance of phytoplankton 


402 



DAYS AFTER 13 JULY 

Figure 25-8. Phytoplankton Cell Counts in the Microcosms and 
in the Lower West Passage of Narragansett Bay During the 
July 1977 Turbulence Experiment. 

NOTE: Zooplankton numbers in the filtered tanks (80 n net) were only slightly 
lower than in the unfiltered (see Figure 25-9). Data points are the means of dup¬ 
licate tanks. 


403 














DAYS AFTER 13 JULY 

Figure 25-9. Total Zooplankton Counts in the Microcosms and in 
the Lower West Passage of Narragansett Bay During the 
July 1977 Turbulence Experiment. 

NOTE: Data points are the mean of duplicate tanks. 


404 


















— BAY 

DAY TURNOVER 
NO PADDLE 
ONE PADDLE 

DAY TURNOVER 
NO PADDLE 
ONE PADDLE 


fO 

O 


E 

— 

D 

O 

O 

< 

_J 

CL 

o 

h- 

>- 

X 

CL 



0 5 10 15 20 25 

DAYS AFTER 3 AUGUST 


Figure 25-10. Counts of Phytoplankton and Total Zooplankton 
in the Microcosms and in the Lower West Passage of Narragansett 
Bay During the August 1977 Turbulence Experiment. 


NOTE: Data points are the mean of duplicate tanks. 


405 











and zooplankton during the warmer months. Unfortunately, the results of the 
experiments do not make it clear if the turbulence effect is felt directly by 
both populations or if the enhancement of phytoplankton growth is the result 
of lower zooplankton grazing pressure in the more turbulent tanks. The lack of 
a significant turbulence effect on phytoplankton during the colder months may 
result from the fact that the phytoplankton and zooplankton virtually do not 
interact at low temperatures when feeding rates and excretion approach zero 
(Heinle and Vargo, 1978). During the warmer months there is evidence from 
some of our other microcosm experiments that the zooplankton are more 
effective at cropping down phytoplankton than a 60 percent artificial level of 
cropping imposed biweekly (Oviatt et al in press). In some cases, such as the 
April run, it appeared that the lower grazing pressure might be due to less 
effective zooplankton feeding as well as to a higher zooplankton mortality in 
the well mixed microcosms. At this point, however, it is still not clear if this 
increased zooplankton mortality was the result of a real physiological or 
behavioral response to the turbulent field or if it was a simple mechanical 
artifact resulting from the manner in which turbulence was generated. 

Not only is the physical basis of turbulence confusing, but, at least at this 
point, so are its ecological consequences. The experiments described here are 
among the first ever reported on this problem, and it is not surprising that so 
much remains obscure. The results demonstrate the potential significance of 
turbulence as an ecological factor in pelagic systems and illustrate the 
importance of carrying out relatively long term (15-30 day) experiments at 
different times of the year, or at least at different temperatures, when studying 
the problem. It is also important to explore different ways of generating 
turbulence as well as the effects of its intensity in experimental ecosystems. 

ACKNOWLEDGEMENTS 

We are grateful to Peter Murphy and Don Winslow of the U.S. 
Environmental Protection Agency, Narragansett, R.I., for their help in 
maintaining the microcosms and in the collection of plankton data. Mark 
Wimbush, Randy Watts, Diego Alonso and Michael Prison of the Graduate 
School of Oceanography at the University of Rhode Island struggled with us 
over the matter of turbulence and its measurement and meaning. Patrick 
Roques and Dana Kester at the Graduate School of Oceanography contributed 
to the gas exchange measurements. The research was supported in part by grant 
No. R-803143 from the U.S.-E.P.A. to the University of Rhode Island. 


406 


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409 


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